CN110623956A - Methods and compositions for killing senescent cells and for treating senescence-associated diseases and disorders - Google Patents

Abstract

Provided herein are methods for selectively killing senescent cells and for treating senescence-associated diseases and disorders by administering a senolytic agent. The senescence-associated diseases and disorders treatable by the methods of using senolytic agents described herein include cardiovascular diseases and disorders associated with or caused by: arteriosclerosis, such as atherosclerosis; idiopathic pulmonary fibrosis; chronic obstructive pulmonary disease; osteoarthritis; aging-related eye diseases and disorders; and aging-related skin diseases and disorders.

Description

Methods and compositions for killing senescent cells and for treating senescence-associated diseases and disorders

The present application is a divisional application of the inventive patent application entitled "methods and compositions for killing senescent cells and for treating senescence-associated diseases and disorders" filed under application No. 201580017167.1, filed on day 2015, 1-month 28, filed on day applicants as barker's aging institute, ewings biotechnology, meiao medical education and research foundation, and john hopkins university.

Statement of government interest

The invention was made with government support under funds from AG009909, AG017242, AG41122 and AG046061 awarded by the national institutes of health. The government has certain rights in this invention.

Statement regarding sequence listing

The sequence listing associated with this application is provided in textual format rather than in paper copy and is incorporated herein by reference. The name of the text file containing the SEQUENCE listing is 200201_419WO _ SEQUENCE _ testing. The text file is 5.2KB, created on day 27/1/2015, and submitted electronically via the EFS-Web.

Background

Technical Field

The disclosure herein generally relates to methods for treating and preventing diseases and disorders associated with aging cells.

Description of the Related Art

Senescent cells accumulate in tissues and organs of an individual as the individual ages and are found in age-related pathological sites. Senescent cells are thought to be important for inhibiting the proliferation of dysfunctional or damaged cells, particularly for arresting the development of malignancies (see, e.g., Campisi, curr. Opin. Genet. Dev.21:107-12 (2011); Campisi, Trends Cell biol.11: S27-31 (2001); Prieur et al, curr. Opin. Cell biol.20: 150-55 (2008)); however, the presence of senescent cells in an individual may contribute to aging and aging-related dysfunction (see, e.g., Campisi, Cell 120:513-22 (2005)). Considering that some aspects of age-related health decline have been attributed to aging cells, and that aging cells can contribute to certain diseases and also be induced as a result of essential life-sustaining chemotherapy and radiation therapy, the presence of aging cells can have deleterious effects on millions of patients worldwide. However, identifying and developing treatments for such diseases and conditions by selectively eliminating aging cells has been a formidable task. The present disclosure satisfies these needs and provides related advantages.

Disclosure of Invention

Provided herein are methods for treating senescence-associated diseases by administering a senolytic agent (senolytic agent). The following are certain embodiments described in more detail herein. As described herein, the senolytic agent is administered in an amount sufficient to selectively kill senescent cells for a time sufficient to selectively kill senescent cells. Also provided herein are methods for selectively killing senescent cells in a subject having a senescence-associated disease or disorder, which in certain embodiments is not cancer, and administering a senolytic agent described herein to a subject in need thereof according to the methods of administration described herein.

In one embodiment, a method is provided for treating a senescence-associated disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a small molecule senolytic agent that selectively kills senescent cells over non-senescent cells; wherein the senescence-associated disease or disorder is not cancer, wherein the senolytic agent is administered in at least two treatment cycles, wherein each treatment cycle independently comprises a treatment course of 1 day to 3 months followed by a non-treatment interval of at least 2 weeks; with the proviso that if the senolytic agent is an MDM2 inhibitor, the MDM2 inhibitor is administered as a monotherapy and the length of each course of treatment is at least 5 days during which the MDM2 inhibitor is administered for at least 5 days. In certain embodiments, the senolytic agent is selected from an MDM2 inhibitor; an inhibitor of one or more BCL-2 anti-apoptotic protein family members, wherein the inhibitor inhibits at least BCL-xL; and specific inhibitors of Akt. In particular embodiments, the MDM2 inhibitor is a cis-imidazoline compound, a spiro-oxindole compound, or a benzodiazepineChemical combinationA compound (I) is provided. In a particular embodiment, the cis-imidazoline compound is a nutlin compound. In particular embodiments, the senolytic agent is an MDM2 inhibitor and is Nutlin-3a or RG-1172. In a particular embodiment, the Nutlin compound is Nutlin-3 a. In particular embodiments, the cis-imidazoline compound is RG-7112, RG7388, RO5503781, or a dihydroimidazothiazole compound. In a particular embodiment, the dihydroimidazothiazole compound is DS-3032 b. In a particular embodiment, the MDM2 inhibitor is a spiro-oxindole compound selected from the group consisting of MI-63, MI-126, MI-122, MI-142, MI-147, MI-18, MI-219, MI-220, MI-221, MI-773, and 3- (4-chlorophenyl) -3- ((1- (hydroxymethyl) cyclopropyl) methoxy) -2- (4-nitrobenzyl) isoindolin-1-one. In particular embodiments, the MDM2 inhibitor is tryptanthrin (serdestemean); a piperidone compound; CGM 097; or an MDM2 inhibitor which also inhibits MDMX and is selected from RO-2443 and RO-5963. In a particular embodiment, the piperidone compound is AM-8553. In particular embodiments, the inhibitor of one or more BCL-2 anti-apoptotic protein family members is a BCL-2/BCL-xL inhibitor; a BCL-2/BCL-xL/BCL-w inhibitor; or a BCL-xL selective inhibitor. In a particular embodiment, the senolytic agent is an inhibitor of one or more BCL-2 anti-apoptotic protein family members, wherein the inhibitor inhibits at least Bcl-xL and is selected from the group consisting of ABT-263, ABT-737, WEHI-539, and A-1155463. In particular embodiments, the BCL-xL selective inhibitor is a benzothiazole-hydrazone compound, an aminopyridine compound, a benzimidazole compound, a tetrahydroquinoline compound, or a phenoxy compound. In a particular embodiment, the benzothiazole-hydrazone compound is a WEHI-539. In particular embodiments, the inhibitor of one or more BCL-2 anti-apoptotic protein family members is A-1155463, ABT-263, or ABT-737. In a particular embodiment, the Akt inhibitor is MK-2206. In particular embodiments, the senolytic agent is an MDM2 inhibitor or an inhibitor of one or more BCL-2 anti-apoptotic protein family members, wherein the inhibitor inhibits at least BCL-xL and is cytotoxic to cancer cells, at each treatment cycleThe total dose of senolytic agent administered in (a) is an amount that is ineffective for treating cancer. In particular embodiments, the senolytic agent is an MDM2 inhibitor or an inhibitor of one or more BCL-2 anti-apoptotic protein family members, wherein the inhibitor at least inhibits BCL-xL and is cytotoxic to cancer cells, and wherein the senolytic agent is administered in two or more treatment cycles wherein the total dose of senolytic agent administered in the two or more treatment cycles is an amount less than a cancer therapeutically effective amount. In particular embodiments, the MDM2 inhibitor is Nutlin-3 a; RG-7112; RG 7388; RO 5503781; DS-3032 b; MI-63; MI-126; MI-122; MI-142; MI-147; MI-18; MI-219; MI-220; MI-221; MI-773; and 3- (4-chlorophenyl) -3- ((1- (hydroxymethyl) cyclopropyl) methoxy) -2- (4-nitrobenzyl) isoindolin-1-one; trademetan; AM-8553; CGM 097; or an MDM2 inhibitor which also inhibits MDMX and is selected from RO-2443 and RO-5963. In particular embodiments, the inhibitor of one or more BCL-2 anti-apoptotic protein family members is ABT-263, ABT-737, A-1155463, or WEHI-539. In another embodiment, the subject has cancer and wherein the senescence-associated disease or disorder is a chemotherapy side effect or a radiation therapy side effect, wherein the senolytic agent is administered to the subject on one or more days beginning on at least the sixth day after the administration cycle of chemotherapy or radiation therapy and not concurrent with chemotherapy or radiation therapy, and wherein the senolytic agent is not a chemotherapeutic agent for treating the cancer, and wherein the senolytic agent is a small molecule and is selected from an MDM2 inhibitor; one or more inhibitors of BCL-2 anti-apoptotic protein family members, wherein the inhibitor inhibits at least BCL-xL and is selected from the group consisting of BCL-2/BCL-xL inhibitors; a BCL-2/BCL-xL/BCL-w inhibitor; and a BCL-xL selective inhibitor; and specific inhibitors of Akt. In another specific embodiment, the chemotherapeutic side effect is selected from the group consisting of gastrointestinal toxicity, peripheral neuropathy, fatigue, malaise, low physical activity, hematologic toxicity, hepatotoxicity, alopecia, pain, mucositis, fluid retention, and skin toxicity. In another specific embodiment, the chemotherapeutic side effect is fatigue. In another particular embodimentIn one embodiment, the side effects of chemotherapy include cardiotoxicity. In another specific embodiment, the senescence-associated disease or disorder is osteoarthritis, atherosclerosis, chronic obstructive pulmonary disease, or idiopathic pulmonary fibrosis. In another specific embodiment, the administration of the senolytic agent comprises three or more treatment cycles. In another specific embodiment, the senolytic agent is administered for one, two, three or four days, provided that the senolytic agent is not an MDM2 inhibitor. In another specific embodiment, the senolytic agent is administered as a monotherapy.

In another embodiment, a method is provided for treating a non-cancer, senescence-associated disease or disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount of a small molecule senolytic agent that selectively kills senescent cells over non-senescent cells and that is cytotoxic to cancer cells, wherein the senolytic agent is administered as a monotherapy over at least one treatment cycle comprising a treatment course followed by a non-treatment interval; and wherein the total dose of senolytic agent administered during the treatment cycle is an amount less than a therapeutically effective amount of cancer, wherein the senolytic agent is (a) an inhibitor of a Bcl-2 anti-apoptotic protein family member that inhibits at least Bcl-xL; (b) MDM2 inhibitors; or (c) an Akt specific inhibitor. In certain embodiments, the senolytic agent is administered in two or more treatment cycles, and wherein the total dose of senolytic agent administered in the two or more treatment cycles is an amount less than a cancer therapeutically effective amount.

In other particular embodiments of the methods described above and herein, each treatment course is no longer than (a) one month, or (b) no longer than two months, or (c) no longer than 3 months. In particular embodiments, each course of treatment is no longer than (a)5 days, (b)7 days, (c)10 days, (d)14 days, or (e)21 days. In particular embodiments, the senolytic agent is administered every two or three days per treatment course. In particular embodiments, the course of treatment is one, two, three or four days. In another specific embodiment, the senolytic agent is administered daily during each course of treatment. In another specific embodiment, the non-treatment interval is at least two weeks, at least one month, at least 2 months, at least 3 months, at least 6 months, at least 9 months, or at least 1 year. In another specific embodiment, the course of treatment is one day. In another specific embodiment, the senescence-associated disease or disorder is a cardiovascular disease selected from atherosclerosis, angina, arrhythmia, cardiomyopathy, congestive heart failure, coronary artery disease, carotid artery disease, endocarditis, coronary thrombosis, myocardial infarction, hypertension, aortic aneurysm, diastolic dysfunction, hypercholesterolemia, hyperlipidemia, mitral valve prolapse, peripheral vascular disease, cardiac stress resistance (cardiac stress resistance), myocardial fibrosis, cerebral aneurysm, and stroke. In another particular embodiment, the senescence-associated disease or disorder is an inflammatory or autoimmune disease or disorder selected from osteoarthritis, osteoporosis, oral mucositis, inflammatory bowel disease, kyphosis, and herniated intervertebral disc. In another particular embodiment, the senescence-associated disease or disorder is a neurodegenerative disease selected from alzheimer's disease, parkinson's disease, huntington's disease, dementia, mild cognitive impairment, and motor neuron dysfunction. In another specific embodiment, the senescence-associated disease or disorder is a metabolic disease selected from diabetes, diabetic ulcer, metabolic syndrome, and obesity. In another specific embodiment, the senescence-associated disease or disorder is a pulmonary disease selected from pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, cystic fibrosis, emphysema, bronchiectasis, and age-related loss of lung function. In another particular embodiment, the senescence-associated disease or disorder is an eye disease or disorder selected from macular degeneration, glaucoma, cataracts, presbyopia, and loss of vision. In another particular embodiment, the senescence-associated disease or disorder is an age-associated disorder selected from renal disease, renal failure, frailty, hearing loss, muscle fatigue, skin condition, skin wound healing, liver fibrosis, pancreatic fibrosis, oral submucosa fibrosis, and sarcopenia (sarcopenia). In another particular embodiment, the senescence-associated disease or disorder is selected from the group consisting of eczema, psoriasis, hyperpigmentation, nevi, rash, atopic dermatitis, urticaria, diseases and disorders associated with photoactivation or photoaging, wrinkles; itching; dysesthesia; outbreak of eczema; -an acidotrophic skin disease; reactive neutrophilic dermatoses; pemphigus; pemphigoid; immune bullous skin disease; proliferation of skin fibroblasts; cutaneous lymphomas; and skin diseases or conditions of cutaneous lupus. In another particular embodiment, the senescence-associated disease or disorder is atherosclerosis; osteoarthritis; pulmonary fibrosis; hypertension or chronic obstructive pulmonary disease. In another specific embodiment, the senolytic agent is administered directly to an organ or tissue comprising the senolytic cell. In another particular embodiment, the senolytic agent is combined with at least one pharmaceutically acceptable excipient to formulate a pharmaceutically acceptable composition to provide timed release of the senolytic agent. In another specific embodiment, the senolytic agent is administered as a bolus infusion. In another specific embodiment, the senescence-associated disease or disorder is osteoarthritis and the senolytic agent is administered directly to the osteoarthritic joint. In another specific embodiment, the senolytic agent is administered intra-articularly to an osteoarthritic joint. In another specific embodiment, the senolytic agent is administered topically, transdermally, or intradermally. In another specific embodiment, the senescence-associated disease or disorder is osteoarthritis and the senolytic agent induces production of type II collagen in a joint. In another specific embodiment, the senescence-associated disease or disorder is osteoarthritis, and the senolytic agent inhibits erosion (erosion) of the proteoglycan layer in the joint. In another specific embodiment, the senescence-associated disease or disorder is osteoarthritis and the senolytic agent inhibits erosion of a bone of a joint. In another specific embodiment, the pulmonary fibrosis is idiopathic pulmonary fibrosis. In another specific embodiment, the senolytic agent reduces the amount of fibrotic lung tissue in the lung. In another specific embodiment, the senolytic agent is administered intranasally, by inhalation, intratracheally, or by intubation. In another specific embodiment, the senescence-associated disease or disorder is atherosclerosis, and wherein the senolytic agent increases the stability of an atherosclerotic plaque. In another particular embodiment, the senescence-associated disease or disorder is atherosclerosis, and wherein the senolytic agent inhibits the formation of atherosclerotic plaques in a blood vessel of the subject. In another particular embodiment, the senescence-associated disease or disorder is atherosclerosis, and wherein the senolytic agent reduces the lipid content of an atherosclerotic plaque in a blood vessel of the subject. In another specific embodiment, the senescence-associated disease or disorder is atherosclerosis, and wherein the senolytic agent increases the fibrous cap thickness of the plaque. In another specific embodiment, the senescent cell is a senescent preadipocyte, a senescent endothelial cell, a senescent fibroblast, a senescent neuron, a senescent epithelial cell, a senescent mesenchymal cell, a senescent smooth muscle cell, a senescent macrophage, or a senescent chondrocyte. In another particular embodiment, the senolytic agent kills at least 20% of the senescent cells and kills no more than 5% of the non-senescent cells in an organ or tissue that comprises senescent cells associated with a senescence-associated disease or disorder. In another particular embodiment, the senolytic agent kills at least 25% of the senescent cells in an organ or tissue that comprises senescent cells associated with a senescence-associated disease or disorder.

In one embodiment, a method is provided for treating osteoarthritis in a subject, the method comprising administering to the subject a therapeutically effective amount of a small molecule senolytic agent that selectively kills senescent cells over non-senescent cells, wherein (a) the senolytic agent is administered in at least two treatment cycles, wherein each treatment cycle independently comprises a treatment course of 1 day to 3 months followed by a non-treatment interval, and wherein the non-treatment interval is at least two weeks; or (b) administering the senolytic agent directly to the osteoarthritic joint. In another specific embodiment, the senolytic agent induces type II collagen production in an osteoarthritic joint. In another specific embodiment, the senolytic agent inhibits the erosion of the proteoglycan layer in an osteoarthritic joint. In another specific embodiment, the senolytic agent inhibits bone erosion of the osteoarthritic joint. Also provided herein in an embodiment is a method for inducing type II collagen production, comprising administering to a subject in need thereof a therapeutically effective amount of a senolytic agent that selectively kills senescent cells over non-senescent cells, wherein (a) the senolytic agent is administered in at least two treatment cycles, wherein each treatment cycle independently comprises a treatment course of 1 day to 3 months followed by a non-treatment interval, wherein the non-treatment interval is at least two weeks; or (b) administering the senolytic agent directly to the osteoarthritic joint. In another specific embodiment, the senolytic agent is administered intra-articularly. In another particular embodiment, the senolytic agent is administered topically, transdermally, or intradermally. In another specific embodiment, the senolytic agent is administered as a bolus infusion. In another particular embodiment, the senolytic agent is combined with at least one pharmaceutical excipient to formulate a pharmaceutical composition that provides timed release of the senolytic agent. In another specific embodiment, the senolytic agent inhibits the erosion of the proteoglycan layer in an osteoarthritic joint. In another specific embodiment, the senolytic agent inhibits bone erosion of the osteoarthritic joint. In another specific embodiment, the senolytic agent kills at least 20% of the senescent cells and kills no more than 5% of the non-senescent cells in the osteoarthritic joint. In another specific embodiment, the senolytic agent kills at least 25% of the senescent cells in the osteoarthritic joint.

In one embodiment, a method is provided for treating a senescence-associated lung disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a small molecule senolytic agent that selectively kills senescent cells over non-senescent cells, wherein the senolytic agent is administered as monotherapy in at least two treatment cycles, wherein each treatment cycle independently comprises a treatment course of 1 day to 3 months followed by a non-treatment interval, wherein the non-treatment interval is at least 2 weeks. In another specific embodiment, a method is provided for treating a senescence-associated lung disease or disorder in a subject, the method comprising administering to the subject a senolytic agent, which is a small molecule compound that selectively kills senescent cells, wherein the senolytic agent is administered in at least two treatment cycles, each cycle comprising a treatment course and a non-treatment interval, and wherein the non-treatment interval is at least 2 months. In a particular embodiment, the senescence-associated lung disease or disorder is pulmonary fibrosis. In another specific embodiment, the pulmonary fibrosis is idiopathic pulmonary fibrosis. In another specific embodiment, the senescence-associated lung disease or disorder is Chronic Obstructive Pulmonary Disease (COPD). In another specific embodiment, the senescence-associated lung disease or disorder is selected from age-related loss of lung function, cystic fibrosis, bronchiectasis, emphysema, and asthma. In another particular embodiment, the senolytic agent is administered directly to the affected lung tissue comprising senescent cells. In another specific embodiment, the senolytic agent is administered by inhalation, intranasally, intratracheally, or by intubation. In another specific embodiment, the senolytic agent is administered as a bolus infusion. In another particular embodiment, the senolytic agent is combined with at least one pharmaceutical excipient to formulate a pharmaceutical composition that provides timed release of the senolytic agent. In another particular embodiment, the senolytic agent kills at least 20% of the senescent cells and kills no more than 5% of the non-senescent cells in the lung of the subject. In another specific embodiment, the senolytic agent kills at least 25% of the senescent cells in the lung of the subject.

In one embodiment, a method is provided for treating a cardiovascular disease or disorder caused by or associated with arteriosclerosis in a subject, the method comprising administering to the subject a therapeutically effective amount of a small molecule senolytic agent that selectively kills senescent cells over non-senescent cells, wherein the senolytic agent is administered in at least two treatment cycles, wherein each treatment cycle independently comprises a treatment course of 1 day to 3 months followed by a non-treatment interval, wherein the non-treatment interval is at least 2 weeks. In particular embodiments, the subject has atherosclerosis, congestive heart failure, peripheral vascular disease, hypertension, or coronary artery disease. In another specific embodiment, the cardiovascular disease or disorder is atherosclerosis. In another particular embodiment, the senolytic agent increases the stability of an atherosclerotic plaque. In another particular embodiment, the senolytic agent reduces the lipid content of an atherosclerotic plaque in a blood vessel of the subject. In another specific embodiment, the senolytic agent increases the fibrous cap thickness of the plaque. In another particular embodiment, the senolytic agent inhibits the formation of an atherosclerotic plaque in a blood vessel of the subject. In another specific embodiment, the likelihood of occurrence of myocardial infarction, angina, stroke, carotid thrombosis, or coronary thrombosis is reduced. In another embodiment, there is provided a method for increasing the stability of an atherosclerotic plaque present in a blood vessel of a subject, the method comprising administering to the subject a therapeutically effective amount of a small molecule senolytic agent that selectively kills senescent cells over non-senescent cells, wherein the senolytic agent is administered in at least two treatment cycles, wherein each treatment cycle independently comprises a treatment course of 1 day to 3 months followed by a non-treatment interval, wherein the non-treatment interval is at least 2 weeks. In particular embodiments, the subject has a cardiovascular disease selected from atherosclerosis, congestive heart failure, peripheral vascular disease, hypertension or coronary artery disease. In another specific embodiment, the cardiovascular disease or disorder is atherosclerosis. In another specific embodiment, the senolytic agent reduces the lipid content of an atherosclerotic plaque in a blood vessel of the subject. In another specific embodiment, the senolytic agent increases the fibrous cap thickness of the plaque. In another particular embodiment, the senolytic agent inhibits the formation of atherosclerotic plaques in a blood vessel of the subject. In another particular embodiment, the senolytic agent reduces the amount of atherosclerotic plaque in a blood vessel of the subject. In another specific embodiment, the senolytic agent is administered parenterally or orally. In another particular embodiment, the senolytic agent is administered directly to an artery comprising senescent cells. In another specific embodiment, the senolytic agent is administered as a bolus infusion. In another particular embodiment, the senolytic agent is combined with at least one pharmaceutical excipient to formulate a pharmaceutical composition that provides timed release of the senolytic agent. In another particular embodiment, the senolytic agent kills at least 20% of the senescent cells and kills no more than 5% of the non-senescent cells in the arteriosclerotic arteries of the subject. In another specific embodiment, the senolytic agent kills at least 25% of the senescent cells in an arteriosclerotic artery of the subject.

In certain embodiments of the methods described above and herein, the treatment course is not longer than one month or not longer than two months. In another specific embodiment, the treatment course is (a) not longer than 5 days, (b) not longer than 7 days, (c) not longer than 10 days, (d) not longer than 14 days, or (e) not longer than 21 days. In another specific embodiment, the senolytic agent is administered every two or three days during the course of treatment. In another specific embodiment, the treatment course is one, two, three or four days. In another specific embodiment, said senolytic agent is administered daily during said treatment course. In another specific embodiment, the non-treatment interval is (a) at least one month, (b) at least 2 months, (c) at least 3 months, (d) at least 6 months, (e) at least 9 months, or (f) at least 1 year. In another specific embodiment, the treatment course is one day and the non-treatment interval is 0.5-12 months. In other particular embodiments, the course of treatment is at least 5 days when the MDM2 inhibitor is administered. In another specific embodiment, the senolytic agent is administered as a monotherapy. In another specific embodiment, the senolytic agent is administered in three or more treatment cycles.

In certain embodiments directed to the methods described above and herein, the senolytic agent is selected from an MDM2 inhibitor; an inhibitor of one or more BCL-2 anti-apoptotic protein family members, wherein the inhibitor inhibits at least BCL-xL; and specific inhibitors of Akt. In another particular embodiment, the MDM2 inhibitor is a cis-imidazoline compound, a spiro-oxindole compound, or a benzodiazepineA compound is provided. In another particular embodiment, the cis-imidazoline compound is a nutlin compound. In another specific embodiment, the Nutlin compound is Nutlin-3 a. In another specific embodiment, the cis-imidazoline compound is RG-7112, RG7388, RO5503781, or a dihydroimidazothiazole compound. In another specific embodiment, the dihydroimidazothiazole compound is DS-3032 b. In another particular embodiment, the MDM2 inhibitor is a spiro-oxindole compound selected from the group consisting of MI-63, MI-126, MI-122, MI-142, MI-147, MI-18, MI-219, MI-220, MI-221, MI-773, and 3- (4-chlorophenyl) -3- ((1- (hydroxymethyl) cyclopropyl) methoxy) -2- (4-nitrobenzyl) isoindolin-1-one. In another particular embodiment, the MDM2 inhibitor is trypticam; a piperidone compound; CGM 097; or an MDM2 inhibitor which also inhibits MDMX and is selected from RO-2443 and RO-5963. In another specific embodiment, the method comprisesThe piperidone compound is AM-8553. In another specific embodiment, the one or more inhibitors of BCL-2 anti-apoptosis protein family members is a BCL-2/BCL-xL inhibitor; a BCL-2/BCL-xL/BCL-w inhibitor; or a BCL-xL selective inhibitor. In another particular embodiment, the BCL-xL selective inhibitor is a benzothiazole-hydrazone compound, an aminopyridine compound, a benzimidazole compound, a tetrahydroquinoline compound, or a phenoxy compound. In another particular embodiment, the benzothiazole-hydrazone compound is a WEHI-539. In another specific embodiment, the inhibitor of one or more BCL-2 anti-apoptotic protein family members is A-1155463, ABT-263, or ABT-737. In another specific embodiment, the Akt inhibitor is MK-2206. In another particular embodiment, when the senolytic agent is a MDM2 inhibitor or an inhibitor of one or more BCL-2 anti-apoptotic protein family members, wherein the inhibitor inhibits at least BCL-xL and is cytotoxic to cancer cells, the total dose of senolytic agent administered in each treatment cycle is an amount that is ineffective for treating cancer. In another specific embodiment, the MDM2 inhibitor is Nutlin-3 a; RG-7112; RG 7388; RO 5503781; DS-3032 b; MI-63; MI-126; MI-122; MI-142; MI-147; MI-18; MI-219; MI-220; MI-221; MI-773; and 3- (4-chlorophenyl) -3- ((1- (hydroxymethyl) cyclopropyl) methoxy) -2- (4-nitrobenzyl) isoindolin-1-one; trademetan; AM-8553; CGM 097; or an MDM2 inhibitor which also inhibits MDMX and is selected from RO-2443 and RO-5963. In another specific embodiment, the inhibitor of one or more BCL-2 anti-apoptotic protein family members is ABT-263, ABT-737, A-1155463, or WEHI-539.

In another embodiment, also provided herein is a method for treating a senescence-associated disease or disorder in a subject, comprising administering to the subject a senolytic agent that is a small molecule MDM2 inhibitor that selectively kills senescent cells over non-senescent cells, wherein the senolytic agent is administered as a monotherapy, wherein the senolytic agent is administered in at least two treatment cycles, wherein each treatment cycle independently comprises treating a senolytic agentA course of treatment followed by a non-treatment interval, wherein the length of the course of treatment is at least 5 days and not longer than three months, during which course of treatment the MDM2 inhibitor is administered for at least 5 days, and wherein the senescence-associated disease or disorder is not cancer. In a particular embodiment, the treatment course is at least 9 days in length. In another specific embodiment, the treatment course is not longer than one month or not longer than two months. In another specific embodiment, the treatment course is no longer than 10, 14 or 21 days. In another particular embodiment, the MDM2 inhibitor is administered daily. In another specific embodiment, the MDM2 inhibitor is administered every two or three days during the course of treatment. In another specific embodiment, the non-treatment interval is at least 2 weeks, at least one month, at least 2 months, at least 6 months, at least 9 months, or at least 1 year. In another specific embodiment, the administration of the MDM2 inhibitor to the subject comprises three or more treatment cycles. In another particular embodiment, the MDM2 inhibitor is a cis-imidazoie compound, a spiro-oxindole compound, or a benzodiazepineA compound is provided. In another specific embodiment, the cis-imidazoline compound is a nutlin compound. In another specific embodiment, the Nutlin compound is Nutlin-3 a. In another specific embodiment, the cis-imidazoline compound is RG-7112, RG7388, or RO5503781, or a dihydroimidazothiazole compound. In another specific embodiment, the dihydroimidazothiazole compound is DS-3032 b. In another particular embodiment, the MDM2 inhibitor is a spiro-oxindole compound selected from the group consisting of MI-63, MI-126, MI-122, MI-142, MI-147, MI-18, MI-219, MI-220, MI-221, MI-773, and 3- (4-chlorophenyl) -3- ((1- (hydroxymethyl) cyclopropyl) methoxy) -2- (4-nitrobenzyl) isoindolin-1-one. In another particular embodiment, the MDM2 inhibitor is trypticam; a piperidone compound; also inhibits MDMX and is selected from the group consisting of RO-2443 and RO-5963A DM2 inhibitor; or CGM 097. In another specific embodiment, the piperidone compound is AM-8553. In another specific embodiment, the method further comprises administering to the subject a small molecule inhibitor of one or more of mTOR, nfk B, PI3-k, and AKT pathways. In another specific embodiment, the method further comprises administering to the subject an Akt-specific inhibitor. In another specific embodiment, the method further comprises the AKT inhibitor is MK-2206.

In one embodiment, a method is provided for treating a senescence-associated disease or disorder in a subject, comprising administering to the subject a senolytic agent that is a small molecule inhibitor of one or more BCL-2 anti-apoptotic protein family members, wherein the inhibitor at least inhibits BCL-XL, wherein the senolytic agent selectively kills senescent cells over non-senescent cells, wherein the senolytic agent is administered in at least two treatment cycles, wherein each treatment cycle independently comprises a treatment course of 1 day to 3 months followed by a non-treatment interval of at least 2 weeks, and wherein the senescence-associated disease or disorder is not a cancer. In another specific embodiment, the inhibitor of one or more BCL-2 anti-apoptotic protein family members is a BCL-2/BCL-xL inhibitor; a BCL-2/BCL-xL/BCL-w inhibitor; or a BCL-xL selective inhibitor. In another particular embodiment, the inhibitor of one or more BCL-2 anti-apoptotic protein family members is a benzothiazole-hydrazone compound, an aminopyridine compound, a benzimidazole compound, a tetrahydroquinoline compound, or a phenoxy compound. In another particular embodiment, the benzothiazole-hydrazone compound is a WEHI-539. In another specific embodiment, the inhibitor of one or more BCL-2 anti-apoptotic protein family members is A-1155463, ABT-263, or ABT-737. In another specific embodiment, the method further comprises administering to the subject a small molecule inhibitor of one or more of mTOR, nfk B, PI3-k, and AKT pathways. In another specific embodiment, the method further comprises administering to the subject an Akt-specific inhibitor. In another specific embodiment, the method further comprises the AKT inhibitor is MK-2206.

In one embodiment, a method is provided for treating a senescence-associated disease or disorder in a subject, comprising administering to the subject a senolytic agent that is a small molecule specific inhibitor of AKT, wherein the senolytic agent selectively kills senescent cells over non-senescent cells, wherein the senolytic agent is administered as a monotherapy in at least two treatment cycles, wherein each treatment cycle independently comprises a treatment course of 1 day to 3 months followed by a non-treatment interval of at least 2 weeks, and wherein the senescence-associated disease or disorder is not a cancer. In another specific embodiment, the AKT inhibitor is MK-2206. In another specific embodiment, the method further comprises administering to the subject a small molecule inhibitor of one or more of the mTOR, nfkb, and PI3-k pathways.

In other particular embodiments of the methods described above and herein, each treatment course is no longer than one month or no longer than two months. In another specific embodiment, each treatment course is (a) no longer than 5 days, (b) no longer than 7 days, (c) no longer than 10 days, (d) no longer than 14 days, or (e) no longer than 21 days. In another specific embodiment, the senolytic agent is administered every two or three days during the course of treatment. In another specific embodiment, each course of treatment is one, two, three or four days. In another specific embodiment, the senolytic agent is administered daily during the treatment course. In another specific embodiment, the non-treatment interval is at least two weeks, one month, at least 2 months, at least 6 months, at least 9 months, or at least 1 year. In another particular embodiment, administering the senolytic agent to the subject comprises three or more treatment cycles. In another specific embodiment, the senolytic agent is administered as a monotherapy. In another particular embodiment, the senescence-associated disease or disorder is a cardiovascular disease selected from atherosclerosis, angina, arrhythmia, cardiomyopathy, congestive heart failure, coronary artery disease, carotid artery disease, endocarditis, coronary thrombosis, myocardial infarction, hypertension, aortic aneurysm, diastolic dysfunction, hypercholesterolemia, hyperlipidemia, mitral valve prolapse, peripheral vascular disease, cardiac stress resistance, myocardial fibrosis, cerebral aneurysm, and stroke. In another specific embodiment, the subject has a cardiovascular disease selected from atherosclerosis, congestive heart failure, peripheral vascular disease, hypertension or coronary artery disease. In another particular embodiment, the senescence-associated disease or disorder is an inflammatory or autoimmune disease or disorder selected from osteoarthritis, osteoporosis, oral mucositis, inflammatory bowel disease, kyphosis, and herniated intervertebral disc. In another particular embodiment, the senescence-associated disease or disorder is a neurodegenerative disease selected from alzheimer's disease, parkinson's disease, huntington's disease, dementia, mild cognitive impairment, and motor neuron dysfunction. In another specific embodiment, the senescence-associated disease or disorder is a metabolic disease selected from diabetes, diabetic ulcer, metabolic syndrome, and obesity. In another particular embodiment, the senescence-associated disease or disorder is a lung disease selected from idiopathic pulmonary fibrosis, chronic obstructive lung disease, asthma, cystic fibrosis, emphysema, bronchiectasis, and age-related loss of lung function. In another particular embodiment, the senescence-associated disease or disorder is an eye disease or disorder selected from macular degeneration, glaucoma, cataracts, and loss of vision. In another specific embodiment, the senescence-associated disease or disorder is an age-associated disorder selected from renal disease, renal failure, frailty, hearing loss, muscle fatigue, skin condition, skin wound healing, liver fibrosis, pancreatic fibrosis, oral submucosa fibrosis, and sarcopenia. In another particular embodiment, the senescence-associated disease or disorder is selected from eczema, psoriasis, hyperpigmentation, nevi, rash, atopic dermatitis, urticaria, diseases and disorders associated with photoallergic or photoaging, wrinkles; itching; dysesthesia; outbreak of eczema; an eosinophilic skin disease; reactive neutrophilic dermatoses; pemphigus; pemphigoid; immune bullous skin disease; skin fiber histiocyte proliferation; cutaneous lymphomas; and skin diseases or conditions of cutaneous lupus. In another specific embodiment, the senescence-associated disease or disorder is atherosclerosis; arthritis of the bone joint; idiopathic pulmonary fibrosis; or chronic obstructive pulmonary disease. In another particular embodiment, the senescence-associated disease or disorder is osteoarthritis and the senolytic agent is administered directly to the osteoarthritic joint. In another specific embodiment, the senolytic agent is administered intra-articularly to an osteoarthritic joint. In another specific embodiment, the senolytic agent is administered topically, transdermally, or intradermally. In another specific embodiment, the senescence-associated disease or disorder is osteoarthritis and the senolytic agent induces production of type II collagen in a joint. In another particular embodiment, the senescence-associated disease or disorder is osteoarthritis, and the senolytic agent inhibits erosion of the proteoglycan layer in the joint. In another particular embodiment, the senescence-associated disease or disorder is osteoarthritis and the senolytic agent inhibits erosion of a bone of a joint. In another particular embodiment, the senescence-associated disease or disorder is idiopathic pulmonary fibrosis, and the senolytic agent reduces the amount of fibrotic lung tissue in the lung. In another specific embodiment, the senolytic agent is administered intranasally, by inhalation, intratracheally, or by intubation. In another particular embodiment, the senolytic agent is combined with at least one pharmaceutically acceptable excipient to formulate a pharmaceutically acceptable composition to provide timed release of the senolytic agent. In another specific embodiment, the senolytic agent is administered as a bolus infusion. In another specific embodiment, the senescence-associated disease or disorder is atherosclerosis, and wherein the senolytic agent increases the stability of an atherosclerotic plaque. In another particular embodiment, the senescence-associated disease or disorder is atherosclerosis, and wherein the senolytic agent inhibits the formation of atherosclerotic plaques in a blood vessel of the subject. In another particular embodiment, the senescence-associated disease or disorder is atherosclerosis, and wherein the senolytic agent reduces the lipid content of an atherosclerotic plaque in a blood vessel of the subject. In another specific embodiment, the senescence-associated disease or disorder is atherosclerosis, and wherein the senolytic agent increases the fibrous cap thickness of the plaque. In another specific embodiment, the senescence-associated disease or disorder is atherosclerosis, and wherein the likelihood of occurrence of myocardial infarction, angina, stroke, carotid thrombosis, or coronary thrombosis is reduced. In another specific embodiment, the senescent cell is a senescent preadipocyte, a senescent endothelial cell, a senescent fibroblast, a senescent neuron, a senescent epithelial cell, a senescent mesenchymal cell, a senescent smooth muscle cell, a senescent macrophage, or a senescent chondrocyte. In another specific embodiment, the senolytic agent kills at least 20% of the senescent cells and kills no more than 5% of the non-senescent cells. In another specific embodiment, the senolytic agent kills at least 25% of senescent cells.

In one embodiment, provided herein is a method for inhibiting metastasis in a subject having a cancer, the method comprising administering to the subject a single small molecule senolytic agent that selectively kills senescent cells over non-senescent cells, wherein the senolytic agent is administered to the subject on one or more days beginning at least the sixth day after an administration cycle of chemotherapy and not concurrent with chemotherapy, and wherein the senolytic agent is not a chemotherapeutic agent for treating the cancer, and wherein the senolytic agent is selected from an MDM2 inhibitor; an inhibitor of one or more BCL-2 anti-apoptotic protein family members, wherein the inhibitor inhibits at least BCL-XL and is selected from a BCL-2/BCL-xL inhibitor; a BCL-2/BCL-xL/BCL-w inhibitor; and a BCL-xL selective inhibitor; and specific inhibitors of Akt. In a particular embodiment, the metastasis is melanoma tumor cellMetastasis of cells, prostate cancer cells, testicular cancer cells, breast cancer cells, brain cancer cells, pancreatic cancer cells, colon cancer cells, thyroid cancer cells, stomach cancer cells, lung cancer cells, ovarian cancer cells, kaposi's sarcoma cells, skin cancer cells, kidney cancer cells, head or neck cancer cells, larynx cancer cells, squamous cancer cells, bladder cancer cells, osteosarcoma cells, cervical cancer cells, endometrial cancer cells, esophageal cancer cells, liver cancer cells, or kidney cancer cells. In another specific embodiment, administering the MDM2 inhibitor to the subject comprises three or more treatment cycles. In another particular embodiment, the MDM2 inhibitor is a cis-imidazoline compound, a spiro-oxindole compound, or a benzodiazepineA compound is provided. In another particular embodiment, the cis-imidazoline compound is a nutlin compound. In another specific embodiment, the Nutlin compound is Nutlin-3 a. In another specific embodiment, the cis-imidazoline compound is RG-7112, RG7388, or RO5503781, or a dihydroimidazothiazole compound. In another specific embodiment, the dihydroimidazothiazole compound is DS-3032 b. In another particular embodiment, the MDM2 inhibitor is a spiro-oxindole compound selected from the group consisting of MI-63, MI-126, MI-122, MI-142, MI-147, MI-18, MI-219, MI-220, MI-221, MI-773, and 3- (4-chlorophenyl) -3- ((1- (hydroxymethyl) cyclopropyl) methoxy) -2- (4-nitrobenzyl) isoindolin-1-one. In another particular embodiment, the MDM2 inhibitor is trypticam; a piperidone compound; an MDM2 inhibitor which also inhibits MDMX and is selected from RO-2443 and RO-5963; or CGM 097. In another specific embodiment, the piperidone compound is AM-8553. In another specific embodiment, the inhibitor of one or more BCL-2 anti-apoptotic protein family members is a BCL-2/BCL-xL inhibitor; a BCL-2/BCL-xL/BCL-w inhibitor; or a BCL-xL selective inhibitor. In another specific embodiment, the inhibitor of one or more BCL-2 anti-apoptotic protein family members is benzeneA benzothiazole-hydrazone compound, an aminopyridine compound, a benzimidazole compound, a tetrahydroquinoline compound, or a phenoxy compound. In another specific embodiment, the benzothiazole-hydrazone compound is a WEHI-539. In another specific embodiment, the inhibitor of one or more BCL-2 anti-apoptotic protein family members is A-1155463, ABT-263, or ABT-737. In another specific embodiment, the senolytic agent is an AKT inhibitor. In yet another specific embodiment, the AKT inhibitor is MK-2206.

In another embodiment, a method for identifying a senolytic agent is provided, the method comprising (a) inducing cellular senescence to provide established senescent cells; (b) contacting a sample of senescent cells with a candidate agent, and contacting a sample of control non-senescent cells with a candidate agent; (c) determining a level of survival of the senescent cells and a level of survival of the non-senescent cells, wherein the candidate agent is a senolytic agent when the level of survival of the senescent cells is less than the level of survival of the non-senescent cells. In particular embodiments, the method further comprises contacting the senolytic agent identified in step (c) with a cell capable of producing collagen; and determining the level of collagen produced by the cells, thereby identifying a senolytic agent for the treatment of osteoarthritis. In a particular embodiment, the cells capable of producing collagen are chondrocytes. In a particular embodiment, the collagen produced is collagen type 2. In particular embodiments, the method further comprises administering the senolytic agent to a non-human animal having a osteoarthritic lesion in a joint and determining one or more of: (a) the level of senescent cells in the joint; (b) somatic functions of the animal; (c) the level of one or more markers of inflammation; (d) histology of the joint; and (e) the level of type 2 collagen produced, thereby determining the therapeutic efficacy of the senolytic agent, wherein one or more of the following is observed in the treated animal compared to an animal not treated with the senolytic agent: (i) a reduction in the level of senescent cells in the joints of the treated animal; (ii) improved somatic function of the treated animal; (iii) a reduction in the level of one or more markers of inflammation in the treated animal; (iv) increased histological normality in the joints of treated animals; and (v) an increase in the level of type 2 collagen produced in the treated animal. In particular embodiments, the method further comprises administering the senolytic agent to a non-human animal of an animal model of atherosclerosis, the animal having atherosclerotic plaques, and determining one or more of: (a) the level of one or more markers of inflammation; and (b) a level of atherosclerotic plaques, thereby determining the therapeutic efficacy of the senolytic agent, wherein one or more of the following is observed in the treated animal compared to an animal not treated with the senolytic agent: (i) a reduction in the level of one or more markers of inflammation in the treated animal; and (ii) a reduction in the level of atherosclerotic plaques in the treated animal; thereby identifying senolytic agents useful for treating atherosclerosis. In particular embodiments, the method further comprises administering the senolytic agent to a non-human animal of an animal model of a pulmonary disease, the animal having pulmonary fibrotic tissue, and determining one or more of: (ii) (a) the level of one or more markers of inflammation; and (b) a level of pulmonary fibrotic tissue, thereby determining the therapeutic efficacy of the senolytic agent, wherein one or more of the following is observed in the treated animal compared to an animal not treated with the senolytic agent: (i) a reduction in the level of one or more markers of inflammation in the treated animal; and (ii) a reduction in the level of pulmonary fibrotic tissue in the treated animal, thereby identifying a senolytic agent for treating a senescence-associated pulmonary disease.

In another embodiment, a method for treating an aging-related disease or disorder in a subject is provided, the method comprising: (a) detecting the level of senescent cells in the subject; and (b) administering to the subject a senolytic agent that selectively kills senescent cells, wherein the senolytic agent is selected from a small molecule and is selected from an MDM2 inhibitor; an Akt specific inhibitor; an inhibitor of one or more BCL-2 anti-apoptotic protein family members, wherein the inhibitor inhibits at least BCL-xL. In particular embodiments, the methods further comprise that the inhibitor of one or more BCL-2 anti-apoptotic protein family members is a Bcl-2/Bcl-xL/Bcl-w inhibitor, a Bcl-2/Bcl-xL inhibitor, a Bcl-xL/Bcl-w inhibitor, or a Bcl-xL selective inhibitor.

In other particular embodiments, there is provided a method for treating, reducing the likelihood of developing, or delaying the onset of a senescent cell-related disease or disorder in a subject having a senescent cell-related disease or disorder or having at least one causative factor for developing a senescent cell-related disease or disorder, the method comprising administering to the subject a senolytic agent that alters one or both of a cell survival signaling pathway and an inflammatory pathway in senescent cells, thereby promoting death of senescent cells, with the proviso that if the subject has cancer, the senolytic agent is not a primary therapy for treating the cancer (primary therapy), wherein the senolytic agent is administered once every 0.5-12 months, and wherein the senescent cell-related disease or disorder is a cardiovascular disease or disorder, An inflammatory disease or disorder, a pulmonary disease or disorder, a neurological disease or disorder, a side effect of chemotherapy, a side effect of radiation therapy, or metastasis. In another particular embodiment, there is provided a method for treating, reducing the likelihood of developing, or delaying the onset of a senescent cell-related disease or disorder in a subject having a senescent cell-related disease or disorder or having at least one causative factor for developing a senescent cell-related disease or disorder, the method comprising administering to the subject a senolytic agent that alters one or both of a cell survival signaling pathway and an inflammatory pathway in senescent cells, thereby promoting death of the senescent cells, wherein the senolytic agent is administered once every 4-12 months.

Also provided herein are uses of the senolytic agents described herein. In one embodiment, there is provided the use of a senolytic agent for the treatment of a senescence-associated disease or disorder, wherein a therapeutically effective amount of a small molecule senolytic agent that selectively kills senescent cells over non-senescent cells is suitable for administration in at least two treatment cycles, wherein each treatment cycle independently comprises a treatment course of 1 day to 3 months followed by a non-treatment interval of at least 2 weeks; with the proviso that if the senolytic agent is an MDM2 inhibitor, the MDM2 inhibitor is administered as a monotherapy and the length of each course of treatment is at least 5 days during which the MDM2 inhibitor is administered for at least 5 days; wherein the senescence-associated disease or disorder is not cancer. The senolytic agents described herein can be used in the manufacture of a medicament for treating a senescence-associated disease or disorder as described herein.

In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, it will be understood by those skilled in the art that the present invention may be practiced without these details. In other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring descriptions of the embodiments. Unless the context requires otherwise, throughout the description and the claims that follow, the word "comprise", and variations such as "comprises" and "comprising", will be interpreted in an open, inclusive sense, i.e., as "including but not limited to". Furthermore, the term "comprising" (and related terms such as "comprises" or "having" or "comprising") is not intended to exclude other certain embodiments, e.g., any constituent embodiment of a substance, composition, method or process, etc., described herein may "consist of" or "consist essentially of" the feature. The headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

Furthermore, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a non-human animal" can refer to one or more non-human animals or a plurality of such animals, and reference to "a cell" or "the cell" includes reference to one or more cells and equivalents thereof (e.g., cells) known to those skilled in the art, and so forth. When steps of a method are described or claimed and are described as occurring in a particular order, the description that a first step occurs (or is performed) "before" (i.e., precedes) a second step has the same meaning if rewritten to describe that the second step occurs (or is performed) "after" the first step. The term "about" when referring to a numerical digit or range of values means that the numerical digit or range of values referred to is an approximation within experimental variation (or within statistical experimental error), and thus the numerical digit or range of values may vary from 1% to 15% of the numerical digit or range of values recited. For example, use of "about X" shall include +/-1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15% of the value X. It should also be noted that the term "or" is generally employed in its sense including "and/or" unless the context clearly dictates otherwise. For example, the term "at least one" when referring to at least one compound or at least one composition has the same meaning and understanding as the term "one or more".

Drawings

FIG. 1 provides treatment of (1) senescence-induced cells by irradiation (Sen (IR)) with Nutlin-3a (nut); (2) senescent-induced cells by doxorubicin treatment (sen (doxo)); and (3) a schematic of a general timeline and procedure for Non-senescent cells (Non Sen).

Figures 2A-D show the effect of Nutlin-3a on the survival rate of age-induced fibroblasts by irradiation. Figure 2A shows the effect on Irradiated (IR) senescent foreskin fibroblasts (sen (IR) HCA2) after 9 days of Nutlin-3a treatment at 0, 2.5 or 10 μ M (D9). Figure 2B shows the percent viability of irradiated BJ cells (sen (ir) BJ) treated with Nutlin 3a at the concentrations indicated. Figure 2C shows the percent survival of irradiated lung fibroblasts (sen (ir) IMR90)), and figure 2D shows the percent survival of irradiated Mouse Embryonic Fibroblasts (MEFs) treated with Nutlin-3 a.

Figures 3A-B show the effect of Nutlin-3A on the survival of cells induced to senescence by doxorubicin treatment. HCA2 cells were treated with Nutlin-3a for 9 days (D9) and aortic endothelial cells were treated with Nutlin-3a for 11 days (D11) and percent survival was then determined. Figure 3A shows the effect of Nutlin-3A on doxorubicin-treated (Doxo) aged foreskin fibroblasts (HCA 2). Figure 3B shows the effect of Nutlin-3a on doxorubicin-treated (Doxo) aged aortic endothelial cells (Endo Aort) (figure 3B).

Figures 4A-C show the percent growth of non-senescent fibroblasts treated with Nutlin-3 a. Cells were treated with Nutlin-3a for 9 days and the percent increase determined (D9). Nutlin-3a is Non-toxic to Non-senescent foreskin fibroblasts (Non Sen hca2) (as shown in fig. 4A), Non-senescent lung fibroblasts (Non Sen IMR90) (as shown in fig. 4B), and Non-senescent lung mouse embryo fibroblasts (Non Sen MEF) (as shown in fig. 4C).

Figures 5A-B show the percent increase in non-senescent aortic endothelial cells and non-senescent preadipocytes treated with Nutlin-3 a. Cells were treated with Nutlin-3a for 11 days and the percent increase determined (D11). Figures 5A and 5B show that Nutlin-3a is Non-toxic to Non-senescent aortic endothelial cells (Non Sen Endo Aort) and Non-senescent preadipocytes (Non Sen Pread), respectively.

Figure 6 shows a schematic of a timeline for treatment and imaging analysis of p16-3MR mice with Nutlin-3 a. On day 35, mice were sacrificed and fat and skin were collected for RNA extraction, and lungs were collected and snap frozen for use in immuno-microscopy. RNA was analyzed for the expression of SASP factors (mmp3, IL-6) and senescence markers (p21, p16 and p 53). DNA damage markers (γ H2AX) were analyzed in frozen lung tissue.

FIG. 7 shows a schematic of the insertion of the p16-3MR transgene. 3MR (trimodal) reporter) is a fusion protein containing the functional domains of synthetic Renilla Luciferase (LUC), monomeric Red fluorescent protein (mRFP) and truncated Herpes Simplex Virus (HSV) -1 thymidine kinase (tTK), which allows for killing by Ganciclovir (GCV). The 3MR cDNA was inserted in-frame (in frame) with p16in exon 2, creating a fusion protein containing the first 62 amino acids of p16, but not the full-length wild-type p16 protein. 3 insertion of MR cDNA also in exon 2 p19^ARFStop codons were introduced in the reading frame.

Figure 8 shows doxorubicin-induced reduction in the luminous intensity of aging in mice. Female C57/Bl6p16-3MR mice were treated with Doxycycline (DOXO). Luminescence was measured after 10 days and used as baseline (100% intensity) for each mouse. Nutlin-3a (nut) (N ═ 9) was administered intraperitoneally daily from day 10 to day 24 after doxorubicin treatment. Luminescence was then measured on days 7, 14, 21, 28, 35 after Nutlin-3a treatment and the final value was calculated as a percentage of the baseline value. Control animals (DOXO) were injected with an equal volume of PBS (N ═ 3).

FIGS. 9A-E show the levels of mRNA for endogenous mmp-3, IL-6, p21, p16, and p53 in skin and fat from animals following treatment with doxorubicin alone (DOXO) or doxorubicin plus Nutlin-3a (DOXO + NUT). The values represent fold induction of a particular mRNA compared to untreated control animals. FIG. 9A: p 21; FIG. 9B: p16^INK4a(p 16); FIG. 9C: p 53; FIG. 9D: mmp-3; and FIG. 9E: IL-6. Data were obtained from doxorubicin-treated mice (Doxo N ═ 3) and dorrubicin + Nutlin-3 a-treated mice (Doxo + Nutlin N ═ 6).

Figures 10A-B show data showing that Nutlin-3a reduces the number of cells with doxorubicin-induced DNA damage. Figure 10A shows immunofluorescence microscopy of lung sections from doxorubicin-treated animals (DOXO) (left panel) and doxorubicin and Nutlin-3 a-treated mice (DOXO + Nutlin) detected by binding to rabbit polyclonal primary antibody specific for γ H2AX, followed by incubation with goat anti-rabbit secondary antibody and subsequent counterstaining with DAPI. Figure 10B shows the percentage of positive cells from immunofluorescence microscopy calculated and expressed as a percentage of the total number of cells. Data were obtained from doxorubicin-treated mice (Doxo N ═ 3) and doxorubicin + Nutlin-3 a-treated mice (Doxo-Nutlin N ═ 3).

Figure 11 shows that Nutlin-3a treatment reduced the senescence-associated (SA) β -galactosidase (β -gal) intensity of fat biopsies from animals first treated with doxorubicin. Female C57/BL6p16-3MR mice were treated with dorbixacin. A portion of doxorubicin-treated animals received Nutlin-3a (nut) or pbs (doxo) daily from day 10 to day 24 after dorbixin treatment. Three weeks after Nutlin-3a treatment, mice were sacrificed and fat biopsies were fixed and stained with a solution containing X-Gal immediately. Untreated animals were used as negative Controls (CTRL).

FIGS. 12A-12C show the detection of IL-6 production in the nuclei of non-senescent (NS) cells and Irradiated (IR) senescent cells treated with Nutlin-3 a. Primary human fibroblasts (IMR90) were irradiated on day-6 and treated with 10 μ M Nutlin-3a or DMSO (vehicle control) in culture medium from day 0 to day 9. Cells were cultured for an additional 6 days (day 12 to day 15) in medium without Nutlin-3a or DMSO. IL-6 in the nuclei of the cells was detected with anti-IL-6 antibody on day 9 and on day 12. The percentage of IL-6 positive nuclei in each of the irradiated Nutlin-3 a-treated cells and DMSO-treated cells is shown in FIG. 12A. The immunofluorescence of cells expressing IL-6 detected with anti-IL-6 antibody is shown in FIG. 12B. FIG. 12C shows the relative levels of IL-6 secretion in senescent cells treated with Nutlin-3a (Sen (IR) Nut3a 10. mu.M) or vehicle (Sen (IR) DMSO) on days 9, 12 and 15 (D9, D12, D15, respectively). Fold increase (fold NS, y-axis) compared to non-senescent cells is shown.

Figures 13A-13F show the levels of senescence-associated proteins (p21, p16, and IL-1a) and SASP factors (CXCL-1, IL-6, and IL-8) expressed by non-senescent (NS) cells and irradiated senescent cells treated with Nutlin-3A. IMR90 cells were irradiated on day-6 and treated with 10 μ M Nutlin-3a or DMSO (vehicle control) in culture medium from day 0 to day 9. Cells were cultured for an additional 6 days (day 12 to day 15) in medium without Nutlin-3a or DMSO, with medium changed on day 12. Quantitative PCR was performed and p21 was detected in non-senescent cells (NS (i.e., day-7)) and in senescent cells treated with Nutlin-3A (sen (ir) Nut3A) or vehicle (sen (ir) DMSO) on days 9 (d9) and 12 (d12) (fig. 13A, p 21/myokinetin, y axis on a logarithmic scale); p16 (FIG. 13B); IL-1a (FIG. 13C); CXCL-1 (FIG. 13D); IL-6 (FIG. 13E); and the level of IL-8 (FIG. 13F) expression. Data are shown relative to expression of myokinesin.

Figure 14 shows an immunoblot that detects the production of protein in senescent cells treated with Nutlin-3 a. IMR90 cells were irradiated on day-6 and treated with Nutlin-3a or DMSO (vehicle control) in culture medium from day 0 to day 9. Cells were cultured for an additional 6 days (day 12 to day 15) in medium without Nutlin-3a or DMSO, and the medium was changed on day 12. The levels of each protein were detected using commercially available antibodies. Data for non-senescent cells (NS) and senescent cells at days 9, 12 and 15 (Xd 9, Xd12 and Xd15, respectively) cultured in 10. mu.M Nutlin-3a (+) or vehicle (-) are shown.

Fig. 15 depicts an exemplary timeline and processing scheme for determining senescent (irradiated cells) and non-senescent cells (non-irradiated cells) for cell counting.

FIG. 16 depicts a graph showing the effect of ABT-263 ("Navi") treatment on Non-senescent IMR90 cells (Non Sen IMR 90).

FIG. 17 depicts a graph showing the effect of ABT-263 treatment on senescent IMR90 cells (Sen (IR) IMR 90).

FIG. 18 depicts the determination of cell viability: ((CTG)) of senescent cells (irradiated cells) and non-senescent cells (non-irradiated cells).

Figure 19 shows a graph showing the effect of ABT-263 treatment on non-senescent and senescent IMR90 cells.

Figure 20 shows a graph showing the effect of ABT-263 treatment on non-senescent and senescent renal epithelial cells.

Figure 21 shows a graph showing the effect of ABT-263 treatment on non-senescent and senescent foreskin fibroblasts (HCA 2).

Figure 22 shows a graph showing the effect of ABT-263 treatment on non-senescent and senescent lung fibroblasts (IMR 90).

Figure 23 shows a graph showing the effect of ABT-263 treatment on non-senescent and pre-senescent adipocytes.

Figure 24 shows a graph showing the effect of ABT-263 treatment on non-senescent and senescent Mouse Embryonic Fibroblasts (MEFs).

FIG. 25 shows a graph showing the effect of ABT-263 treatment on non-senescent and senescent smooth muscle cells (Smth Mscl).

Figure 26 shows a graph showing the effect of ABT-199 treatment on non-senescent and senescent IMR90 cells.

Figure 27 shows a graph showing the effect of ABT-199 treatment on non-senescent and senescent IMR90 cells.

Figure 28 shows a graph showing the effect of olbacra (Obatoclax) treatment on non-senescent and senescent IMR90 cells.

Fig. 29A and 29B: FIG. 29A shows a graph showing the effect of ABT-263(Navi) treatment in combination with 10nM MK-2206 on non-senescent and senescent IMR90 cells. FIG. 29B shows the percent survival of non-senescent IMR90 cells (IMR90NS) and senescent IMR90 cells (IMR90Sen (IR)) when exposed to MK-2206 alone.

Fig. 30A-B show the effect of WEHI-539 on the percent survival of senescent irradiated lung fibroblasts (sen (ir) IMR90)) (fig. 30A) and irradiated kidney cells (sen (ir)) (fig. 30B). NS is a non-senescent cell that has not been exposed to radiation.

FIG. 31 shows that the senolytic activity of WEHI-539 is inhibited in the presence of a caspase inhibitor (pan caspase inhibitor, Q-VD-OPh). The effect of WEHI-539 on killing senescent cells (IMR90Sen (IR)) is shown on the left side of FIG. 31. The data points within the boxed area depict the killing of senescent cells at WEHI-539 concentrations of 1.67 μ M and 5 μ M to which non-senescent cells (NS) and senescent cells (Sen (IR)) were exposed in the presence or absence of Q-VC-OPh. The percentage survival of non-senescent and senescent cells in the presence and absence of pan-caspase inhibitor (Q-VD in the figure) is shown in the lower right of the figure.

Figure 32 shows the effect of specific shRNA molecules on the survival of senescent cells. Senescent and non-senescent IMR90 cells are transduced with lentiviral vectors comprising shRNA molecules specific for each of the polynucleotides encoding BCL-2, BCL-xL, and BCL-w. The ratio of senescent cell viability to non-senescent cell viability for each shRNA is shown. Each bar represents the average of triplicates. The shRNA sequences introduced into the cells were as follows (left to right): BCL-2: 1,3,3, 5; BCL-XL: 7,9,11, 13; BCL-w: 15,17,19, 21; two non-transduced (NT) samples.

Fig. 33 shows the effect of ABT-737 on the viability of non-senescent lung fibroblasts (IMR90) (IMR90NS) and senescent lung fibroblasts (IMR90) (IMR90Sen (IR)).

FIG. 34 shows that in the presence of a caspase inhibitor (pan caspase inhibitor, Q-VD-OPh), the senolytic activity of ABT-263 is inhibited. The effect of ABT-263 on killing senescent cells (IMR90Sen (IR)) is shown on the upper left of figure 34. Non-senescent cells (NS) and senescent cells (Sen (IR)) were exposed to ABT-263 at concentrations of 0.33. mu.M and 1. mu.M in the presence or absence of the pan-caspase inhibitor Q-VC-OPh. The percent survival of non-senescent and senescent cells in the presence and absence of pan-caspase inhibitor (Q-VD in the figure) is shown in the lower right of FIG. 34.

Figure 35 depicts an animal study design used to assess the efficacy of senescent cell removal by Nutlin-3A treatment (in C57BL6/J mice) or by GCV treatment (in 3MR mice) in inhibiting the signs and progression of osteoarthritis. Group 1 animals (16x C57BL6/J mice; 1x 3MR mice) represent an Anterior Cruciate Ligament (ACL) control group that underwent surgery to remove the ACL of one hind limb (ACL surgery or osteoarthritis surgery (OA)) to induce osteoarthritis. Group 1 animals received an intra-articular injection of vehicle (10 μ Ι) daily for 5 days during the 2 nd postoperative period, and an optional second treatment period at the 4 th postoperative period, concurrent with GCV treatment in the test animals. Group 2 animals (3x 3MR mice) represent a treatment group that received ACL surgery and received daily intra-articular injections of GCV (2.5 μ g/joint) for 5 days during the 2 nd postoperative period and an optional second treatment period at the 4 th postoperative period. Group 3 animals (12x C57BL6/J) represent the second treatment group, which received ACL surgery and received intraarticular injections of Nutlin-3A (5.8 μ g/joint) every other day for 2 weeks starting at week 3 post-surgery. Group 4 animals represent a second control group, which underwent a sham surgery without ACL truncation, and received daily intra-articular injections (10 μ Ι) of vehicle for 5 days during postoperative period 2, and an optional second treatment cycle at postoperative period 4, in parallel with GCV-treated 3MR mice. The study design, such as the amount and schedule of administration (e.g., days) may be adapted for other senolytic agents.

Figure 36 depicts a timeline for the animal study design described in figure 35.

Figures 37A-C show the levels of senescence-associated proteins (p16) and SASP factors (IL-6 and MMP13) expressed by cells from the joints of mice that underwent osteoarthritis surgery (OA surgery), the joints of mice that underwent OA surgery and received Nutlin-3A treatment (Nutlin-3A), the joints of joints that underwent sham surgery and the joints of control mice that did not undergo any surgery (control). Quantitative PCR was performed and p16 was detected in cells extracted from the joints of mice that underwent OA surgery, mice that underwent OA surgery and Nutlin-3A treatment, mice that received sham surgery and controls (no surgery) (fig. 37A); IL-6 (FIG. 37B); and levels of MMP13 (fig. 37C) expression. Data are shown relative to actin expression. The data show that Nutlin-3A treatment eliminates senescent cells from the joint.

Figure 38 shows the level of collagen type 2 expressed by cells from the joints of mice that underwent osteoarthritis surgery (OA surgery), the joints of mice that underwent OA surgery and received Nutlin-3A treatment (Nutlin-3A), the joints that received sham surgery, and the joints of control mice that did not receive any surgery. Quantitative PCR was performed, and the level of collagen type 2 in cells extracted from the joints of mice subjected to OA surgery, mice subjected to OA surgery and Nutlin-3A treatment, mice subjected to sham surgery and control (no surgery) mice was detected. Data are shown relative to actin expression. The data show that Nutlin-3A treatment drives the de novo production of collagen in OA joints.

Figure 39 shows the balance (incapacitiance) measurements taken 4 weeks after osteoarthritis surgery by testing the weight bearing test of which leg the mouse prefers to use. The mice were placed in the compartment and stood on each scale with 1 hind paw. The weight placed on each hind limb was then measured over a period of 3 seconds. At least 3 separate measurements were made for each animal at each time point and the results were expressed as a percentage of the weight placed on the operated limb/the weight placed on the contralateral non-operated limb.

FIG. 40 depicts the results of the weight bearing test shown in FIG. 39. Osteoarthritis results in mice preferring non-operated legs over operated legs (Δ). Clearing senescent cells with Nutlin-3A avoids this effect (. v.).

Figure 41 depicts the results of a hotplate analysis to provide an assessment of sensitivity and response to a painful stimulus. Paw licking (Paw-lick) response time (measured in seconds) in the postoperative limb resulting from reaching pain threshold after placement on a 55 ℃ platform was measured 4 weeks after Osteoarthritis (OA) surgery. The data show that Nutlin-3A treatment shortened OA surgical mice compared to untreated OA surgical mice (■)The reaction time of (2).

Fig. 42 shows a sample from an animal that was not treated by surgery (no surgery (C57B)); animals that received surgery for osteoarthritic and vehicle (OA surgery (3 MR)); and histopathological results of animals subjected to OA surgery and treated with Nulin-3a (OA surgery + Nutlin-3 a). The arrow points to the intact or disrupted proteoglycan layer in the joint.

FIGS. 43A-B show LDLR fed on a High Fat Diet (HFD)^-/-Schematic representation of two animal model studies of atherosclerosis in transgenic mice. The study shown in FIG. 43A evaluated the use of an aging scavenger (e.g., Nutlin-3A) from LDLR^-/-Plaque removal in miceThe extent to which the aging cells reduce plaque burden. The study shown in FIG. 43B evaluated secondary LDLR^-/-(iii) degree to which ganciclovir-based senescent cell depletion ameliorates pre-existing atherogenic disease in/3 MR dual transgenic mice.

FIGS. 44A-D depict LDLR-^/Graph of plasma lipid levels in mice. FIG. 44A shows LDLR-^/In contrast, LDLR-^/-total cholesterol level in mice. FIG. 44B shows LDLR-^/In contrast, LDLR-^/HDL levels in mice. FIG. 44C shows LDLR-^/In contrast, LDLR-^/Triglyceride levels in mice. FIG. 44D shows LDLR-^/In contrast, LDLR-^/vLDL/LDL/IDL levels in mice.

FIGS. 45A-D show LDLR-^/RT-PCR analysis of SASP factor and aging markers in the aortic arch of mice. Fig. 45A shows the aortic arch (within the box). FIGS. 45B-C show expression levels of SASP factor and senescence marker normalized to GAPDH and expressed as age-matched LDLR-^/Fold change in mice. FIG. 45D shows the data of FIGS. 45B-C in digital form.

FIGS. 46A-C show LDLR-^/RT-PCR analysis of SASP factor and aging markers in the aortic arch of mice. FIGS. 46A-B show expression levels of SASP factor and senescence marker normalized to GAPDH and expressed as age-matched LDLR-^/Fold change in mice. Fig. 46C shows the data of fig. 46A-B in digital form.

FIGS. 47A-C show LDLR-^/Staining analysis of aortic plaques in mice. Fig. 47A shows the aorta. Figure 47B shows the percentage of aorta coverage in the plaque. Figure 47C shows sudan iv (sudan iv) staining of the aorta to visualize plaques and represent the area covered by lipid plaques in each sample as a percentage of the total surface area of the aorta.

FIGS. 48A-B depict LDLR-^/Graphs of platelet counts (fig. 48A) and lymphocyte counts (fig. 48B) of mice.

FIGS. 49A-B depict LDLR-^/Graph of body weight and body fat/lean tissue composition (%) in mice.

FIG. 50 depicts LDLR-^/-and LDLR-^/Graph of the effect of more coxivir on the clearance of senescent cells in/3 MR mice, as measured by the percentage of aorta covered in plaques.

FIG. 51 depicts LDLR-^/-and LDLR-^/Graph of the effect of more coxivir on the clearance of senescent cells in/3 MR mice, as measured by the plaque cross-sectional area of the aorta.

FIG. 52 shows the effect of senescent cell clearance on resistance to cardiac stress with aging. AP20187 was injected three times a week in FVB x 129Sv/E x C57BL/6 mixed or C57BL/6 pure genetic background 12 month old INK-ATTAC transgenic mice (mixed group 0.2mg/kg and C57BL/6 group 2mg/kg, respectively). At 18 months, a subset of male and female mice from each group were subjected to cardiac stress testing and the time to cardiac arrest was recorded. The control group received an injection of vehicle.

FIG. 53 shows an RT-PCR analysis of Sur2a expression in female INK-ATTAC transgenic mice described in FIG. 52.

FIGS. 54A-C show LDLR-^/-/3MR double transgenic mice and LDLR-^/Staining analysis of aortic plaques in control mice. FIGS. 54A-B show Sudan IV staining of the aorta to separately LDLR-^/Control mice and LDLR-^/Visualization of plaques in-/3 MR mice. Figure 54C shows the percentage of aorta covered in the plaque as measured by sudan IV stained area.

FIGS. 55A-D show LDLR-^/-/3MR double transgenic mice and LDLR-^/Plaque morphological analysis of control mice. FIGS. 55A and C show Sudan IV staining of the aorta to separately LDLR-^/Control mice and LDLR-^/Visualization of plaques in-/3 MR mice. Circled plaques were harvested and cut into sections and stained to characterize the general architecture of atherosclerotic plaques (fig. 55B and D). "#" indicates fat located outside the aorta.

Figure 56 shows that SA- β -GAL crystals are restricted to lipid-bearing foam cells from atherosclerotic arteries of mice fed a high fat diet. Macrophage foam cells are shown by white dashed outline lines, and adjacent to macrophage foam cells are smooth muscle foam cells. The area in the left box in macrophage foam cells was enlarged and shown on the upper right to show lysosomes with SA- β -GAL crystals. The area in the box inside the smooth muscle foam cells is enlarged and shown on the lower right side of the figure.

Figure 57 shows macrophage foam cells from atherosclerotic arteries of mice fed a high fat diet. Lipid-bearing lysosomes containing SA- β -GAL crystals are indicated by arrows. Figure 58 shows that SA- β -GAL crystals are localized to lysosomes of smooth muscle foam cells in the atherosclerotic arteries of mice fed a high fat diet. The region in the box in the lower left part of the drawing is enlarged and displayed in the upper left inset.

FIG. 59 shows senescent cell clearance versus peripheral capillary oxygen saturation (SpO) in bleomycin-exposed mice₂) The influence of (c).

Figures 60A-C show the effect of senescent cell clearance with ganciclovir on lung function in bleomycin-exposed 3MR mice. Figure 60A shows the effect of ganciclovir treatment on lung elasticity in 3MR mice exposed to bleomycin. Figure 60B shows the effect of ganciclovir treatment on dynamic lung compliance (compliance) in 3MR mice exposed to bleomycin. Figure 60C shows the effect of ganciclovir treatment on static lung compliance in bleomycin-exposed 3MR mice.

FIG. 61 shows senescent cell clearance versus peripheral capillary oxygen saturation (SpO) in mice after 2 and 4 months of exposure to Cigarette Smoke (CS)₂) The influence of (c). AP 20187; GAN ═ ganciclovir; navi ═ Navitoclax (ABT-263); and Nutlin ═ Nutlin 3A.

Figure 62 shows the effect of RG-7112 (structure shown at the top of figure 62) on the percent survival of senescent irradiated lung fibroblast cells IMR90((IMR90) sen (ir)) and non-senescent IMR90 cells, which were not exposed to radiation after 3 days of treatment (lower left) and six days of treatment with RG-7112 (lower right).

FIGS. 63A-B show that paclitaxel induces senescence in p16-3MR mice. Groups of mice were treated with 20mg/kg paclitaxel or vehicle three times every two days (n-4). The luminescence levels in mice treated with paclitaxel are shown in fig. 63A. For the target gene in animals treated with paclitaxel: each of p16, 3MR transgene, and IL-6, the levels of mRNA in skin were determined, as shown in figure 63B.

FIG. 64 shows the effect of ABT-263 on mice initially treated with paclitaxel. Schematic representations of experiments performed in 3MR mice are shown on the right of the figure. Mice were first treated with paclitaxel and then vehicle, ganciclovir (gcv) or ABT-263. Against paclitaxel + vehicle (pacli + vehicle); paclitaxel + ganciclovir (pacli + gcv); rotawheel counts (Wheel counts) were measured for each group of paclitaxel + ABT-263 (pacli + ABT-263) treated mice (n ═ 4) and for control animals that did not receive paclitaxel (see figure 64, left panel).

Fig. 65 shows the effect of treatment with chemotherapeutic agents: thalidomide (100 mg/kg; 7 injections per day); romidepsin (1 mg/kg; 3 injections); pomalidomide (5 mg/kg; 7 injections per day); lenalidomide (50 mg/kg; 7 injections per day); levels of aging induced in the p16-3MR group of animals (n-4) treated with 5-azacytidine (5 mg/kg; 3 injections) and doxorubicin (10 mg/kg; 2-4 injections per week). Measuring the level of luminescence in animals treated with the drug.

Figure 66 shows an immunoblot showing the levels of different cellular proteins in aged and non-aged human abdominal subcutaneous preadipocytes. Senescence was induced as described in example 28. Lysates were prepared at several time points after induction of senescence and the levels of each protein in the lysates were assayed at 24 hours and on days 3,5, 8, 11, 15, 20, and 25 (D3, D5, D8, D11, D15, D20, and D25).

Fig. 67 shows that the group of p16-3MR mice (n ═ 6) fed with a high fat diet (high fat) for four months had an increased number of senescent cells compared to the mice (n ═ 6) fed with a regular food diet (food feeding).

Figure 68 shows a reduction of senescent cells in adipose tissue of p16-3MR mice fed a high fat diet for four months and subsequently treated with ganciclovir compared to vehicle-treated mice. The presence of senescent cells in perirenal, epididymal (Epi) or subcutaneous inguinal (ingg) adipose tissue was detected by SA- β -Gal staining.

Figures 69A-C show the effect of ganciclovir treatment on glucose tolerance in p16-3MR mice fed a high fat diet. A bolus of glucose was given at time zero and blood glucose was monitored for a maximum of 2 hours to determine the efficacy of glucose utilization (fig. 69A). This was quantified as the area under the curve (AUC), with a higher AUC indicating glucose intolerance. The Glucose Tolerance Test (GTT) AUC of mice treated with ganciclovir is shown in figure 69B. Hemoglobin A1C is shown in fig. 69C. n is 9; ANOVA.

Figures 70A-70B show insulin sensitivity (insulin tolerance test (ITT)) of p16-3MR mice fed a high fat diet following ganciclovir administration. Blood glucose levels were measured at 0, 14, 30, 60 and 120 minutes after the glucose bolus administration at time zero (see fig. 70A). No change was observed in the insulin tolerance test when ganciclovir was administered to wild-type mice (see figure 70B).

Figure 71 shows the effect of a-1155463 on the percent survival of senescent irradiated lung fibroblasts (sen (ir) IMR90)) and the percent survival of non-senescent IMR90 cells (sen (ir)). NS is a non-senescent cell that has not been exposed to radiation.

Detailed Description

Aging is a risk factor for most chronic diseases, disability and decline in health. Senescent cells, cells that cease to replicate, accumulate as an individual ages and can partially or significantly cause cellular and tissue deterioration that causes aging and age-related diseases. Cells may also become senescent after exposure to environmental, chemical or biological insults or due to disease. Provided herein are methods and agents for selectively killing senescent cells associated with a variety of pathologies and diseases, including age-related pathologies and diseases. As disclosed herein, diseases and disorders associated with senescent cells can be treated or prevented (i.e., reduced in likelihood of occurrence or development) by administering at least one senolytic agent. Diseases or disorders associated with aging cells that are treated or prevented by the agents and methods described herein include cardiovascular diseases or disorders, inflammatory or autoimmune diseases or disorders, pulmonary diseases or disorders, neurological diseases or disorders, skin diseases or disorders, chemotherapy side effects, radiation therapy side effects, or metastasis or metabolic diseases, all of which are described in more detail herein. In certain particular embodiments, the senescent cell-associated diseases or disorders treated or prevented by the senolytic agents and methods described herein include, for example, Idiopathic Pulmonary Fibrosis (IPF), Chronic Obstructive Pulmonary Disease (COPD), osteoarthritis, and cardiovascular diseases and disorders associated with arteriosclerosis, such as atherosclerosis. In certain embodiments, the senescence-associated disease or disorder is not cancer. As described in more detail herein, senolytic agents include, for example, MDM2 inhibitors (e.g., nutlin 3a, RG-7112); one or more inhibitors of a BCL-2 anti-apoptotic protein family member that inhibits at least the function of the anti-apoptosis protein BCL-xL (e.g., ABT-263, ABT-737, WEHI-539, A-1155463); and Akt-specific inhibitors (e.g., MK-2206).

The senolytic agents described herein are sufficient to kill a significant number of senescent cells. Although cells continue to become senescent in treated subjects, establishment of senescence occurs over several days as shown by the presence of the senescence-associated secretory phenotype (SASP) (see, e.g., Laberge et al, Aging Cell 11:569-78 (2012); Coppe et al, PLoS Biol 6:2853-68 (2008); Coppe et al PLoS One5:39188 (2010); Rodier et al, nat. Cell Biol.11: 973-979; Freund et al, EMBO J.30: 1536-1548 (2011)). Thus, the use of the senolytic agents described herein provides the advantage that these agents can be administered less frequently, intermittently, and/or at lower doses than many therapeutic agents commonly used to treat these diseases and conditions. The methods described herein describe the use of agents, such as senolytic agents, that can be administered less frequently, intermittently, and/or at lower doses than when the agents are used to treat cancer or other diseases.

Aging scavenger

A senolytic agent as used herein is an agent that "selectively" (preferentially or to a greater extent) destroys, kills, removes or promotes selective destruction of senescent cells. In other words, a senolytic agent destroys or kills senescent cells in a biologically, clinically, and/or statistically significant manner as compared to its ability to destroy or kill non-senescent cells. The senolytic agent is used in an amount and for a time sufficient to selectively kill established senescent cells, but insufficient to kill (destroy, cause death) non-senescent cells in a clinically significant or biologically significant manner. In certain embodiments, a senolytic agent described herein alters at least one signaling pathway in a manner that induces (initiates, stimulates, triggers, activates, promotes) and causes (i.e., causes) senescent cell death. For example, by antagonizing proteins within cell survival and/or inflammatory pathways in senescent cells, senolytic agents can alter, for example, one or both of cell survival signaling pathways (e.g., Akt pathways) or inflammatory pathways.

Without wishing to be bound by a particular theory, the mechanism by which the inhibitors and antagonists described herein selectively kill senescent cells is by inducing (activating, stimulating, removing inhibition of apoptotic pathways that cause cell death) apoptotic pathways that cause cell death. The non-senescent cells may be proliferating cells or may be quiescent cells. In certain instances, exposure of non-senescent cells to a senolytic agent as used in the methods described herein can temporarily reduce the ability of the non-senescent cells to proliferate; however, apoptotic pathways were not induced and non-senescent cells were not destroyed.

Certain senolytic agents that can be used in the methods described herein have been described as useful for treating cancer; however, in methods for treating senescence-associated disorders or diseases, senolytic agents are administered in a manner that would be considered different and likely ineffective for treating cancer. Methods for treating a senescence-associated disease or disorder with a senolytic agent described herein can include one or more of reducing the daily dose, reducing the cumulative dose within a single treatment cycle, or reducing the cumulative dose of the agent from multiple treatment cycles as compared to the dose of the agent required for cancer treatment; thus, the likelihood of occurrence of one or more adverse effects (i.e., side effects) associated with treating a subject according to a regimen optimized for treating cancer is reduced. Conversely, as a senolytic agent, a compound described herein can be administered at a lower dose as currently described in the art or in a manner that selectively kills senescent cells (e.g., intermittent administration). In certain embodiments, the senolytic agents described herein can be administered at a lower cumulative dose per treatment course or cycle that will likely be insufficiently cytotoxic to cancer cells to effectively treat the cancer. In other words, according to the methods described herein, the senolytic agent is not used in a manner understood by those skilled in the art as the first choice therapy for treating a cancer, whether the agent is administered alone or in conjunction with one or more additional chemotherapeutic agents or radiation therapy to treat the cancer. Although the agents used in the methods described herein are not used in a manner sufficient to be considered a first choice cancer therapy, the methods described herein and senolytic combinations can be used in a manner useful for inhibiting metastasis (e.g., short course of treatment). As used herein, "first line therapy of cancer" means that when an agent, which may be used alone or with one or more agents, is intended or known to be an effective treatment for cancer (as determined by one of skill in the medical and oncology arts), the administration regimen for treating the cancer with the agent has been designed to achieve the relevant cancer-related objective. To further reduce toxicity, senolytic agents can be administered at a site proximal to or in contact with the senescent cells (non-tumor cells). Local delivery of senolytic agents is described in more detail herein.

The senolytic agents described herein alter (i.e., interfere with, affect) one or more cellular pathways that are activated during the senescence process of a cell. Senolytic agents can alter a cell survival signaling pathway (e.g., Akt pathway) or an inflammatory pathway, or both a cell survival signaling pathway and an inflammatory pathway, in senescent cells. Activation of certain cellular pathways during senescence reduces or inhibits the ability of the cell to induce and eventually undergo apoptosis. Without wishing to be bound by theory, the mechanism by which senolytic agents selectively kill senescent cells is by inducing (activating, stimulating, abrogating inhibition of apoptotic pathways that cause cell death) apoptotic pathways that cause cell death. By interacting with one, two or more target proteins in one or more pathways, senolytic agents can alter one or more signaling pathways in the senescent cell, which results in the elimination or reduction of inhibition of cell death pathways, such as apoptotic pathways. Contacting or exposing senescent cells to senolytic agents to alter one, two or more cellular pathways in the senescent cells may restore the cells' mechanisms and pathways that initiate apoptosis. In certain embodiments, a senolytic agent is an agent that alters a signaling pathway in a senescent cell, which in turn inhibits secretion and/or expression of one or more gene products important for the survival of the senescent cell. Senolytic agents can inhibit the biological activity of a gene product important for the survival of senescent cells. Alternatively, a decrease or decrease in the level of a gene product in a senescent cell may alter the biological activity of another cellular component, which triggers, activates or stimulates the apoptosis pathway or abrogates or decreases the inhibition of the apoptosis pathway. As described herein, a senolytic agent is a biologically active agent and is capable of selectively killing senescent cells in the absence of linkage or conjugation to a cytotoxic moiety (e.g., a toxin or a cytotoxic peptide or a cytotoxic nucleic acid). The senolytic agent is also active in selectively killing senescent cells in the absence of linkage or conjugation to a targeting moiety (e.g., an antibody or antigen-binding fragment thereof; a cell-binding peptide) that selectively binds to senescent cells.

Two alternative modes of cell death can be distinguished: apoptosis and necrosis. Kerr and coworkers (Br. J. cancer26:239-57(1972)) first described the phenomenon as a pattern of cell death morphologically distinct from coagulative necrosis using the term apoptosis. Apoptosis is generally characterized by Cell rounding, chromatin condensation (nuclear pyknosis), nuclear fragmentation (nuclear rupture) and engulfment by neighboring cells (see, e.g., Kroemer et al, Cell Death differ.16:3-11 (2009)). Several molecular assays have been developed and used in the art; however, morphological changes detected by light microscopy and electron microscopy are considered in the art as the best techniques to distinguish between two distinct modes of cell death (see, e.g., Kroemer et al, supra). Alternative modes of cell death, such as caspase-independent apoptosis-like Programmed Cell Death (PCD), autophagy, necrosis-like PCD, and mitotic disorders are also characterized (see, e.g., Golstein, biochem. Sci.32:37-43 (2007); Leist et al, nat. Rev. mol. cell biol.2:589-98 (2001)). See, e.g., Caruso et al, Rare Tumors5(2): 68-71 (2013); published online on 6.7.2013.doi: 10.3081/rt.2013.e 18. Techniques and methods described herein and routinely practiced in the art (e.g., TUNEL) can be used to demonstrate that contact with the senolytic scavenger described herein causes apoptotic cell death.

In certain embodiments, the senolytic agent used in the methods as described herein is a small molecule compound. These senolytic agents that are small molecules may also be referred to herein as senolytic compounds. In certain embodiments, the small molecule senolytic agent comprises an activated senolytic agent or a senolytic agent that is a prodrug that is converted to an active form by an intracellular enzyme. In a more specific embodiment, the enzyme that converts the prodrug into an active senolytic form is an enzyme that is expressed at a higher level in senescent cells than in non-senescent cells.

Senolytic agents described herein that can alter at least one signaling pathway include agents that inhibit the activity of at least one target protein within the pathway. The senolytic agent can be a specific inhibitor of one or more BCL-2 anti-apoptotic protein family members, wherein the inhibitor inhibits at least BCL-xL (e.g., a Bcl-2/Bcl-xL/Bcl-w inhibitor; a selective Bcl-xL inhibitor; a Bcl-xL/Bcl-w inhibitor); inhibitors specific for Akt kinase; or an MDM2 inhibitor. In embodiments, molecules such as quercetin (and analogs thereof), enzastarin, and dasatinib are excluded and are not compounds used in the methods and compositions described herein. In other particular embodiments, the methods comprise the use of at least two senolytic agents, wherein at least one senolytic agent and the second senolytic agent are each different and independently alter one or both of a resident signaling pathway and an inflammatory pathway in the senescent cell.

Small molecules

Senolytic agents that can be used in a method for treating or preventing a senescence-associated disease or disorder include small organic molecules. Small organic molecules (also referred to herein as small molecules or small molecule compounds) typically have less than 10⁵Dalton, less than 10⁴Dalton or less than 10³Molecular weight in daltons. In certain embodiments, the small molecule senolytic agent does not violate the following criteria more than one time: (1) no more than 5 hydrogen bond donors (total number of nitrogen-hydrogen and oxygen-hydrogen bonds); (2) no more than 10 hydrogen bond acceptors (all nitrogen or oxygen atoms); (3) molecular weight less than 500 daltons; (4) octanol-water partition coefficient [5]log P is not greater than 5.

MDM2 inhibitors

In certain embodiments, the senolytic agent can be an MDM2 inhibitor. Can be used for selectively killing senescent cells and treating or preventing senescence-associated diseases or disorders (i.e., reducing or diminishing senescence-associated diseasesLikelihood of occurrence or development of a disease or disorder) the MDM2 (murine double minute 2) inhibitor used in the method of treatment may be a small molecule compound belonging to any one of the following compound species: for example, cis-imidazoline compounds, spiro-oxindole compounds, benzodiazepinesCompounds, piperidone compounds, tryptamine compounds and CGM097 and related analogs. In certain embodiments, the MDM2 inhibitor is also capable of binding to and inhibiting the activity of MDMX (murine double minute X, which is also known as HDMX in humans). The human homolog of MDM2 is known in the art as HDM2 (human double minute 2). Thus, when the subject treated by the methods described herein is a human subject, a compound described herein, such as an MDM2 inhibitor, also inhibits binding of HDM2 to one or more of its ligands.

MDM2 is described in the art as an E3 ubiquitin ligase that can promote tumor formation by targeting tumor suppressor proteins such as p53 for proteasomal degradation by the 26S proteasome (see, e.g., Haupp et al Nature 387: 296-19 91997; Honda et al FEBS Lett 420: 25-27 (1997); Kubbutat et al Nature 387: 299-303 (1997)). MDM2 also affects p53 by binding directly to the N-terminus of p53, thereby inhibiting the transcriptional activation function of p53 (see, e.g., Momand et al, Cell 69: 1237-1245 (1992); Oliner et al, Nature 362: 857-860 (1993)). p53 in turn regulates Mdm 2; the p53 response element is located in the promoter of the Mdm2 gene (see, e.g., Barak et al, EMBO J12: 461-68 (1993)); juven et al, Oncogene 8: 3411-16 (1993); perry et al, Proc.Natl.Acad.Sci.90: 11623-27 (1993)). The existence of such a negative feedback loop between p53 and Mdm2 has been demonstrated by single cell studies (see, e.g., Lahav, exp.med.biol.641: 28-38 (2008)). See also Manfredi, Genes & Development 24:1580-89 (2010).

Several activities and biological functions of MDM2 have been reported. These reported activities include the following: as ubiquitin ligase E3 against itself and ARRB 1; allowing nuclear output of p 53; promoting proteasome-dependent, ubiquitin-independent degradation of retinoblastoma RB1 protein; inhibition of DAXX-mediated apoptosis by induction of ubiquitination and degradation thereof; a component of the TRIM28/KAP1-MDM2-p53 complex involved in stabilizing p 53; a component of the TRIM28/KAP1-ERBB4-MDM2 complex that links a growth factor to a DNA damage response pathway; mediate ubiquitination and subsequent proteasomal degradation of DYRK2 in the nucleus; ubiquitinate IGF1R and SNAI1 and facilitate their proteasomal degradation. MDM2 has also been reported to induce monoubiquitination of the transcription factor FOXO4 (see, e.g., Brenkman et al, PLOS One 3(7): e2819, doi:10.1371/journal. po. 0002819). The MDM2 inhibitors described herein may disrupt the interaction between MDM2 and any one or more of the above cellular components.

In one embodiment, the compounds useful in the methods described herein are cis-imidazoline small molecule inhibitors. Cis-imidazoline compounds include compounds known in the art as nutlin. Like other MDM2 inhibitors described herein, nutlin is a cis imidazoline small molecule inhibitor of the interaction between MDM2 and p53 (see vasillev et al, Science 303 (5659): 844-48 (2004)). Exemplary cis-imidazoline compounds that can be used in methods of selectively killing senescent cells and treating or preventing (i.e., reducing or diminishing the likelihood of occurrence or development of) a senescence-associated disease or disorder are described in U.S. patent nos. 6,734,302, 6,617,346, 7,705,007 and U.S. patent application publication nos. 2005/0282803, 2007/0129416, 2013/0225603. In certain embodiments, the methods described herein comprise the use of a Nutlin compound referred to as Nutlin-1; or a Nutlin compound known as Nutlin-2; or a Nutlin compound designated Nutlin-3 (see CAS registry numbers 675576-98-4 and 548472-68-0). The active enantiomer of Nutlin-3(4- [ [4S,5R) -4, 5-bis (4-chlorophenyl) -4, 5-dihydro-2- [ 4-methoxy-2- (1-methylethoxy) phenyl ] -1H-imidazol-1-yl ] carbonyl ] -2-piperazinone) is known in the art as Nutlin-3 a. In certain embodiments, the methods described herein comprise the use of Nutlin-3a to selectively kill senescent cells.

Nutlin-3 is described in the art as a non-genotoxic activator of the p53 pathway, and activation of p53 is under the control of the murine double minute 2(MDM2) gene. MDM2 protein is an E3 ubiquitin ligase and controls p53 half-life through ubiquitin-dependent degradation. Treatment of certain cancers (e.g., retinoblastoma) by Nutlin-3a was studied in preclinical studies (e.g., for pediatric cancers) and clinical trials. To date, in vitro and preclinical studies using Nutlin-3 have shown that the compound has variable biological effects on the function of cells exposed to the compound. For example, Nutlin-3 has been reported to increase the extent of cancer apoptosis in hematological malignancies, including B cell malignancies (see, e.g., Zauli et al, clin. cancer res.17:762-70 (2011; published on line at 11/24/2010) and references cited therein), and to have a synergistic cytotoxic effect when combined with other chemotherapeutic drugs, such as dasatinib (see, e.g., Zauli et al, supra).

Another exemplary cis-imidazoline small molecule compound for selectively killing senescent cells is RG-7112(Roche) (CAS number 939981-39-2; IUPAC name: ((4S,5R) -2- (4- (tert-butyl) -2-ethoxyphenyl) -4, 5-bis (4-chlorophenyl) -4, 5-dimethyl-4, 5-dihydro-1H-imidazol-1-yl) (4- (3- (methylsulfonyl) propyl) piperazin-1-yl) methanone see U.S. Pat. No. 7,851,626; Tovar et al, Cancer Res.72:2587-97 (2013)).

In another particular embodiment, the MDM2 inhibitor is a cis imidazoline compound known as RG7338 (Roche) (IPUAC name: 4- ((2R,3S,4R,5S) -3- (3-chloro-2-fluorophenyl) -4- (4-chloro-2-fluorophenyl) -4-cyano-5-neopentylpyrrolidine-2-carboxamido) -3-methoxybenzoic acid) (CAS 1229705-06-9); ding et al, J.Med.chem.56 (14) 5979-83.Doi: 10.1021/jm400487c.Epub.2013, 7 months and 16 days; zhao et al, j.med.chem.56(13):5553-61(2013) doi:10.1021/jm4005708.epub 2013, 6 months and 20 days). Another exemplary nutlin compound is RO 5503781. Other useful cis-imidazoline small molecule compounds include dihydroimidazothiazole compounds (e.g., DS-3032 b; Daiichi Sankyo) described by Miyazaki (see, e.g., Miyazaki et al, bioorg. med. chem. lett. 23(3):728-32 (2013)) doi: 10.1016/j.bmcl.2012.11.091.epub.2012, 12/1, Miyazaki et al, bioorg.med. chem. lett.22(20):6338-42 (2012) doi:10.1016/j.bmcl.2012.08.086.epub2012, 8/30, international patent application publication No. WO 2009/151069 (2009)).

In other embodiments, the cis-imidazoline compound that can be used in the methods described herein is a dihydroimidazothiazole compound.

In other embodiments, the MDM2 small molecule inhibitor is a spiro-oxindole compound. See, e.g., in Ding et al, j.am.chem.soc.2005; 127: 10130-31; shangary et al, Proc Natl Acad Sci USA 2008; 105: 3933-38; shangary et al, Mol Cancer Ther 2008; 1533-42 parts of; shangary et al, Mol Cancer Ther 2008; 1533-42 parts of; hardcastle et al, bioorg.Med.chem.Lett.15: 1515-20 (2005); hardcastle et al, J.Med.chem.49(21):6209-21 (2006); watson et al, bioorg.Med.chem.Lett.21(19):5916-9(2011) doi:10.1016/j.bmcl.2011.07.084. Epub.2011, 8/9. Other examples of spiro-oxindole compounds that are MDM2 inhibitors are known in the art as MI-63, MI-126, MI-122, MI-142, MI-147, MI-18, MI-219, MI-220, MI-221, and MI-773. Another specific spiro-oxindole compound is 3- (4-chlorophenyl) -3- ((1- (hydroxymethyl) cyclopropyl) methoxy) -2- (4-nitrobenzyl) isoindolin-1-one. Another compound is known as MI888 (see, e.g., Zhao et al, J.Med.chem.56(13):5553-61 (2013); International patent application publication No. WO 2012/065022).

In other embodiments, the MDM2 small molecule inhibitor that may be used in the methods described herein is a benzodiazepineDiketones (see, e.g., Grasberger et al, J Med Chem 2005; 48: 909-12; Parks et al, Bioorg Med Chem Lett 2005; 15: 765-70; Raboisson et al, Bioorg. Med. Chem. Lett.15:1857-61 (2005); Koblissh et al, mol. cancer ther.5:160-69 (2006)). Benzodiazepines useful in the methods described hereinThe diketone compound includes 1, 4-benzodiazepine-2, 5-diketones. BenzodiazepineExamples of the diketone compound include 5- [ (3S) -3- (4-chlorophenyl) -4- [ (R) -1- (4-chlorophenyl) ethyl]-2, 5-dioxo-7-phenyl-1, 4-diaza-1-yl]Valeric acid and 5- [ (3S) -7- (2-bromophenyl) -3- (4-chlorophenyl) -4- [ (R) -1- (4-chlorophenyl) ethyl]-2, 5-dioxo-1, 4-diaza-1-yl]Valeric acid (see, e.g., Raboisson et al, supra). Other benzodiazepinesThe diketone compound is known in the art as TDP521252(IUPAC name: 5- [ (3S) -3- (4-chlorophenyl) -4- [ (1R) -1- (4-chlorophenyl) ethyl]-7-ethynyl-2, 5-dioxo-3H-1, 4-benzodiazepine-1-yl]Valeric acid) and TDP665759(IUPAC name: (3S) -4- [ (1R) -1- (2-amino-4-chlorophenyl) ethyl]-3- (4-chlorophenyl) -7-iodo-1- [3- (4-methylpiperazin-1-yl) propyl]-3H-1, 4-benzodiazepines2, 5-diketones) (see, e.g., Parks et al, supra; koblish et al, supra) (Johnson&Johnson,New Brunswick,NJ)。

In yet another embodiment, the MDM2 small molecule inhibitor is terphenyl (see, e.g., Yin et al, Angew Chem Int Ed Engl 2005; 44: 2704-. In yet another specific embodiment, the MDM2 inhibitor that may be used in the methods described herein is quilinol (see, e.g., Lu et al, J Med Chem 2006; 49: 3759-62). In yet another specific embodiment, the MDM2 inhibitor is a chalcone (see, e.g., Stoll et al, Biochemistry 2001; 40: 336-44). In yet another specific embodiment, the MDM2 inhibitor is a sulfonamide (e.g., NSC279287) (see, e.g., Galatin et al, J Med Chem 2004; 47: 4163-65).

In other embodiments, the compounds that can be used in the methods described herein are tryptamine, such as Trametan (JNJ-26854165; chemical name: N1- (2- (1H-indol-3-yl) ethyl) -N4- (pyridin-4-yl) benzene-1, 4-diamine; CAS number 881202-45-5) (Johnson & Johnson, New Brunswick, NJ). Tryptophan is a tryptamine derivative that activates p53 and acts as an HDM2 ubiquitin ligase antagonist (see, e.g., Chargari et al, Cancer Lett.312(2):209-18(2011) doi:10.1016/j. canlet.2011.08.011. Epub.2011.8/22 days; Kojima et al, mol. Cancer Ther.9:2545-57 (2010); Yuan et al, J.Hematol. Oncol.4:16 (2011)).

In other particular embodiments, MDM2 small molecule inhibitors that may be used in the methods described herein include those described in Rew et al, j.med.chem.55:4936-54 (2012); Gonzalez-Lopez de turkio et al, j.med.chem.56:4053-70 (2013); sun et al, J.Med.chem.57:1454-72 (2014); gonzalez et al, j.med. chem.2014 4 months [ electronic plate prior to printing plate ]; those described in Gonzalez et al, j.med. chem.2014, 6 months [ electronic plate precedes printing plate ].

In other embodiments, the MDM2 inhibitor is a piperidone compound. An example of a potent MDM2 piperidone inhibitor is AM-8553({ (3R,5R,6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1- [ (2S,3S) -2-hydroxy-3-pentyl ] -3-methyl-2-oxo-3-piperidinyl } acetic acid; CAS number 1352064-70-0) (Amgen, Thousand Oaks, California).

In other particular embodiments, the MDM2 inhibitor that can be used in the methods described herein is piperidine (Merck, Whitehouse Station, NJ) (see, e.g., international patent application publication No. WO 2011/046771). In other embodiments, the MDM2 inhibitor that may be used in the methods is an imidazole-indole compound (Novartis) (see, e.g., international patent application publication No. WO 2008/119741).

Examples of compounds that bind to MDM2 and MDMX and that can be used in the methods described herein include RO-2443 and RO-5963((Z) -2- (4- ((6-chloro-7-methyl-1H-indol-3-yl) methylene) -2, 5-dioxoimidazolidin-1-yl) -2- (3, 4-difluorophenyl) -N- (1, 3-dihydroxypropan-2-yl) acetamide) (see, e.g., Graves et al, proc.natl.acad.sci.usa 109:11788-93 (2012); see, e.g., Zhao et al, 2013, BioDiscovery, supra).

In another particular embodiment, an MDM2 inhibitor, referred to in the art as CGM097, can be used in the methods described herein to selectively kill senescent cells and treat senescence-associated diseases or disorders.

Inhibitors of the BCL-2 family of anti-apoptotic proteins

In certain embodiments, the senolytic agent can be an inhibitor of one or more proteins in the BCL-2 family. In certain embodiments, the at least one senolytic agent is selected from an inhibitor of one or more BCL-2 anti-apoptotic protein family members, wherein the inhibitor inhibits at least BCL-xL. Inhibitors of the BCL-2 family of anti-apoptotic proteins alter at least cell survival pathways. Activation of apoptosis can occur via an exogenous pathway triggered by activation of cell surface death receptors or an endogenous pathway triggered by developmental cues and various intracellular stresses. This endogenous pathway, also known as the stress pathway or mitochondrial pathway, is primarily regulated by the BCL-2 family, a key regulator of caspase activation that consists of: anti-apoptotic (pro-survival) proteins having a BH1-BH4 domain (BCL-2 (i.e., a BCL-2 protein member of the BCL-2 anti-apoptotic protein family), BCL-xL, BCL-w, A1, MCL-1, and BCL-B); pro-apoptotic proteins with BH1, BH2, and BH3 domains (BAX, BAK, and BOK); and pro-apoptotic BH3 protein only (pro-apoptotic BH3-only protein) (BIK, BAD, BID, BIM, BMF, HRK, NOXA, and PUMA) (see, e.g., Cory et al, Naturereviews Cancer 2:647-56 (2002); Cory et al, Cancer Cell 8:5-6 (2005); Adams et al, Oncogene 26: 1324-. BCL-2 anti-apoptotic proteins block activation of the pro-apoptotic multi-domain proteins BAX and BAK (see, e.g., Adams et al, Oncogene 26:1324-37 (2007)). Although the exact mechanism of apoptosis regulation is unknown, it is postulated that only the BH3 protein released by intracellular stress signals binds to the anti-apoptotic BCL-2-like protein via the BH3 "ligand" of the "receptor" BH3 binding groove formed by the BH1-3 region on the anti-apoptotic protein, thereby neutralizing the anti-apoptotic protein (see, e.g., Letai et al, Cancer Cell 2:183-92 (2002); Adams et al, Oncogene, supra). BAX and BAK can subsequently form oligomers in the mitochondrial membrane, leading to membrane permeabilization, cytochrome C release, caspase activation, and ultimately apoptosis (see, e.g., Adams et al, Oncogene, supra).

As used herein and unless otherwise indicated, BCL-2 family members inhibited by the agents described herein are pro-survival (anti-apoptotic) family members. The senolytic agent used in the methods described herein inhibits one or more functions of the BCL-2 anti-apoptotic protein BCL-xL (which may also be written as BCL-xL, or BCL-xL herein and in the art). In certain embodiments, in addition to inhibiting BCL-xL function, the inhibitor may also inhibit and/or interact with one or more functions of BCL-2 (i.e., BCL-xL/BCL-2 inhibitors). In yet another specific embodiment, the senolytic agent used in the methods described herein is classified as an inhibitor of each of BCL-xL and BCL-w (i.e., a BCL-xL/BCL-w inhibitor). In another particular embodiment, senolytic agents used in the methods described herein that inhibit BCL-xL may also inhibit and interact with one or more functions of each of BCL-2 (i.e., BCL-2 protein) and BCL-w (i.e., BCL-xL/BCL-2/BCL-w inhibitor), resulting in selective killing of senescent cells. In certain embodiments, the BCL-2 anti-apoptotic protein inhibitor interferes with the interaction between a BCL-2 anti-apoptotic protein family member (including at least BCL-xL) and one or more ligands or receptors that would bind to the BCL-2 anti-apoptotic protein family member in the absence of the inhibitor. In other particular embodiments, the inhibitor of one or more BCL-2 anti-apoptotic protein family members (wherein the inhibitor inhibits at least BCL-xL) specifically binds only to one or more of BCL-xL, BCL-2, BCL-w, and does not specifically bind to other Bcl-2 anti-apoptotic Bcl-2 family members such as Mcl-1 and BCL2A 1.

In another embodiment, the senolytic agent used in the methods described herein is a BCL-xL selective inhibitor and inhibits one or more functions of BCL-xL. Such senolytic agents that are selective inhibitors of BCL-xL do not inhibit the function of one or more other BCL-2 anti-apoptotic proteins in a biologically or statistically significant manner. BCL-xL may also be referred to herein and in the art as BCL2L1, BCL 2-like 1, BCLX, BCL2L, BCLxL, or BCL-X. In one embodiment, the BCL-xL selective inhibitor alters (e.g., reduces, inhibits, attenuates, prevents) one or more functions of BCL-xL, but does not significantly inhibit one or more functions of other proteins in the BCL-2 anti-apoptotic protein family (e.g., BCL-2 or BCL-w). In certain embodiments, a BCL-xL selective inhibitor interferes with the interaction between BCL-xL and one or more ligands or receptors that would bind BCL-xL in the absence of the inhibitor. In certain particular embodiments, a senolytic agent that inhibits one or more functions of BCL-xL selectively binds to human BCL-xL and does not selectively bind to other proteins in the BCL-2 family to achieve selective killing of senescent cells.

BCL-xL is an anti-apoptotic member of the BCL-2 protein family. BCL-xL also plays an important role in crosstalk (crosstalk) between autophagy and apoptosis (see, e.g., Zhou et al, FEBS j.278:403-13 (2011)). BCL-xL also appears to be involved in bioenergy metabolism (including mitochondrial ATP production, Ca²⁺Flux and protein acetylation) and in several other cellular and biological processes such as mitosis, platelet aggregation and synaptic efficiency (see, e.g., Michels et al, International Journal of Cell Biology, vol.2013, article ID 705294, page 10, 2013.doi: 10.1155/2013/705294). In certain embodiments, a BCL-xL inhibitor described herein can disrupt the interaction between BCL-xL and any one or more of the BH3-only proteins described above to promote apoptosis.

In certain embodiments, the BCL-xL inhibitor is a selective inhibitor, meaning that it preferentially binds to BCL-xL over other anti-apoptotic BCL2 family members (e.g., BCL-2, MCL-1, BCL-w, BCL-b, and BFL-1/A1). In certain embodiments, the BCL-XL selective inhibitor exhibits at least 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold, 10000-fold, 20000-fold, or 30000-fold selectivity for binding to a BCL-XL protein or nucleic acid relative to a BCL-2 protein or nucleic acid. In certain embodiments, the BCL-XL selective inhibitor exhibits at least 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold, 10000-fold, 20000-fold, or 30000-fold selectivity for binding to a BCL-XL protein or nucleic acid relative to the MCL-1 protein or nucleic acid. In certain embodiments, the BCL-XL selective inhibitor exhibits at least 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold, 10000-fold, 20000-fold, or 30000-fold selectivity for binding to a BCL-XL protein or nucleic acid relative to a BCL-w protein or nucleic acid. In certain embodiments, the BCL-XL selective inhibitor exhibits at least 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold, 10000-fold, 20000-fold, or 30000-fold selectivity for binding to a BCL-XL protein or nucleic acid relative to a BCL-B protein or nucleic acid. In certain embodiments, the BCL-XL selective inhibitor exhibits at least 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold, 10000-fold, 20000-fold, or 30000-fold selectivity for binding to a BCL-XL protein or nucleic acid relative to a1 protein or nucleic acid. As described herein, in certain embodiments, the inhibitor of one or more BCL-2 anti-apoptotic protein family members does not detectably bind to MCL-1 or BCL2a1, wherein the inhibitor at least inhibits BCL-xL (e.g., a BCL-xL selective inhibitor).

Methods for measuring the binding affinity of a BCL-xL inhibitor for a BCL-2 family protein are known in the art. For example, the binding affinity of a BCL-xL inhibitor can be determined using a competitive fluorescence polarization assay in which a fluorescent BAK BH3 domain peptide is incubated with a BCL-xL protein (or other BCL-2 family protein) as previously described in the presence or absence of an increased concentration of a BCL-XL inhibitor (see, e.g., U.S. patent publication No. 20140005190; Park et al, Cancer Res.73:5485-96 (2013); Wang et al, Proc. Natl. Acad. Sci USA 97:7124-9 (2000));zhang et al, anal. biochem.307:70-5 (2002); bruncko et al, J.Med.chem.50:641-62 (2007)). The percent inhibition can be determined by the following formula: 1- [ (well mP value-negative control)/Range)]x 100%. The inhibition constant (K) was determined by the following equation_i) The value of (c): k_i＝[I]₅₀/([L]₅₀/K_d+[P]₀/K_d+1) as described in Bruncko et al, J.Med.chem.50:641-62(2007) (see also Wang, FEBS Lett. 360:111-114 (1995)).

Agents that selectively kill senescent cells (e.g., BCL-xL selective inhibitors, BCL-xL/BCL-2/BCL-w inhibitors, BCL-xL/BCL-w inhibitors) for use in the methods described herein include, for example, small molecules.

In particular embodiments, the BCL-xL inhibitor is a small molecule compound belonging to the following classes of compounds: such as any of benzothiazole-hydrazone compounds, aminopyridine compounds, benzimidazole compounds, tetrahydroquinoline compounds, and phenoxy compounds, and related analogs.

In one embodiment, the BCL-xL selective inhibitors useful in the methods described herein are benzothiazole-hydrazone small molecule inhibitors. Benzothiazole-hydrazone compounds include WEHI-539 (5- [3- [4- (aminomethyl) phenoxy ] propyl ] -2- [ (8E) -8- (1, 3-benzothiazol-2-ylhydrazono) -6, 7-dihydro-5H-naphthalen-2-yl ] -1, 3-thiazole-4-carboxylic acid), which is a BH3 peptidomimetic that selectively targets BCL-xL (see, e.g., Lessene et al, Nature chemical biology 9: 390-. In certain embodiments, the methods described herein comprise using WEHI-539 to selectively kill senescent cells.

In other embodiments, the BCL-xL selective inhibitor is an aminopyridine compound. An aminopyridine compound that may be used as a selective BCL-xL inhibitor is BXI-61(3- [ (9-amino-7-ethoxyacridin-3-yl) diazenyl ] pyridine-2, 6-diamine) (see, e.g., Park et al, Cancer Res.73:5485-96 (2013); U.S. patent publication No. 2009-0118135). In certain embodiments, the methods described herein comprise the use of BXI-61 to selectively kill senescent cells.

In other embodiments, the BCL-xL selective inhibitors that can be used in the methods described herein are benzimidazole compounds. An example of a benzimidazole compound that can be used as a selective BCL-XL inhibitor is BXI-72(2 '- (4-hydroxyphenyl) -5- (4-methyl-1-piperazinyl) -2, 5' -bis (1H-benzimidazole) trihydrochloride) (see, e.g., Park et al, supra). In certain embodiments, the methods described herein comprise the use of BXI-72 to selectively kill senescent cells.

In yet another embodiment, the BCL-xL selective inhibitor is a tetrahydroquinoline compound (see, e.g., U.S. patent publication No. 2014-. Examples of tetrahydroquinoline compounds that can be used as selective BCL-xL inhibitors are shown and described in Table 1 of U.S. patent publication No. 2014-0005190. In addition to BCL-xL, other inhibitors described therein can inhibit other BCL-2 family members (e.g., BCL-2).

In other embodiments, the BCL-xL selective inhibitor is a phenoxy compound. An example of a phenoxy compound that can be used as a selective BCL-xL inhibitor is 2[ [3- (2, 3-dichlorophenoxy) propyl ] amino ] ethanol (2,3-DCPE) (see Wu et al, Cancer Res.64:1110-1113 (2004)). In certain embodiments, the methods described herein comprise the use of 2,3-DCPE to selectively kill senescent cells.

In another embodiment, inhibitors of Bcl-2 anti-apoptotic family members that inhibit at least BCL-xL are described in U.S. Pat. No. 8,232,273. In particular embodiments, the inhibitor is a BCL-xL selective inhibitor referred to as A-1155463 (see, e.g., Tao et al, ACS Med. chem. Lett.,2014,5(10): 1088-.

In other embodiments, the senolytic agent of interest also inhibits other BCL-2 anti-apoptotic family members other than BCL-xL. For example, the methods described herein include the use of BCL-xL/BCL-2 inhibitors, BCL-xL/BCL-2/BCL-w inhibitors, and BCL-xL/BCL-w inhibitors and the like. In certain embodiments, the inhibitor comprises a compound that inhibits BCL-2 and BCL-xL, which inhibitor also inhibits BCL-w. Examples of such inhibitors include ABT-263 (4- [4- [ [2- (4-chlorophenyl) -5, 5-dimethylcyclohexen-1-yl ] methyl ] piperazin-1-yl ] -N- [4- [ [ (2R) -4-morpholin-4-yl-1-phenylthiobutan-2-yl ] amino ] -3- (trifluoromethylsulfonyl) phenyl ] sulfonyl benzamide or IUPAC, (R) -4- (4- ((4 '-chloro-4, 4-dimethyl-3, 4,5, 6-tetrahydro- [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) -N- ((4- ((4-morpholinyl-1- (phenylthio) phenyl) Yl) butan-2-yl) amino) -3- ((trifluoromethyl) sulfonyl) phenyl) sulfonyl) benzamide) (see, e.g., Park et al, 2008, j.med.chem.51: 6902; tse et al, Cancer res, 2008,68: 3421; international patent application publication No. WO 2009/155386; U.S. Pat. Nos. 7390799,7709467, 7906505, 8624027), and ABT-737(4- [4- [ (4 '-chloro [1,1' -biphenyl ] -2-yl) methyl ] -1-piperazinyl ] -N- [ [4- [ [ (1R) -3- (dimethylamino) -1- [ (phenylthio) methyl ] propyl ] amino ] -3-nitrophenyl ] sulfonyl ] benzamide, 4- [4- [ (4 '-chloro [1,1' -biphenyl ] -2-yl) methyl ] -1-piperazinyl ] -N- [ [4- [ [ (1R) -3- (dimethylamino) -1- [ (phenylthio) methyl ] propyl ] amino ] -3-nitrophenyl ] sulfonyl ] -or 4- [4- [ [2- (4-chlorophenyl) phenyl ] methyl ] piperazin-1-yl ] -N- [4- [ [ (2R) -4- (dimethylamino) -1-phenylsulfanylbutan-2-yl ] amino ] -3-nitrophenyl ] sulfonylbenzamide) (see, e.g., Oltersdorf et al, Nature,2005,435: 677; U.S. patent nos. 7973161; U.S. patent No. 7642260). In other embodiments, the BCL-2 anti-apoptotic protein inhibitor is a quinazoline sulfonamide compound (see, e.g., sleeps et al, 2011, j.med.chem.54: 1914). In another embodiment, the BCL-2 anti-apoptotic protein inhibitor is a small molecule compound as described in Zhou et al, j.med. chem.2012, 55:4664 (see, e.g., compound 21 (R) -4- (4-chlorophenyl) -3- (3- (4- (4- (4- ((4- (dimethylamino) -1- (phenylthio) butan-2-yl) amino) -3-nitrophenylsulfonylamino) phenyl) piperazin-1-yl) phenyl) -5-ethyl-1-methyl-1H-pyrrole-2-carboxylic acid) and Zhou et al, j.med.chem.2012, 55:6149 (see, e.g., compound 14(R) -5- (4-chlorophenyl) -4- (3- (4- (4- (4-chlorophenyl) - (4- ((4- (dimethylamino) -1- (phenylthio) butan-2-yl) amino) -3-nitrophenylsulfonylamino) phenyl) piperazin-1-yl) phenyl) -1-ethyl-2-methyl-1H-pyrrole-3-carboxylic acid; the compound 15(R) -5- (4-chlorophenyl) -4- (3- (4- (4- (4- ((4- (dimethylamino) -1- (phenylthio) butan-2-yl) amino) -3-nitrophenylsulfonylamino) phenyl) piperazin-1-yl) phenyl) -1-isopropyl-2-methyl-1H-pyrrole-3-carboxylic acid). In other embodiments, the BCL-2 anti-apoptotic protein inhibitor is a BCL-2/BCL-xL inhibitor such as BM-1074 (see, e.g., Aguilar et al, 2013, j.med.chem.56: 3048); BM-957 (see, e.g., Chen et al, 2012, j.med. chem.55: 8502); BM-1197 (see, e.g., Bai et al, PLoS One2014Jun 5; 9(6): e99404.Doi: 10.1371/journal. bone. 009904); U.S. patent application No. 2014/0199234; n-acyl sulfonamide compounds (see, e.g., International patent application publication No. WO 2002/024636, International patent application publication No. WO 2005/049593, International patent application publication No. WO 2005/049594, U.S. Pat. No. 7767684, U.S. Pat. No. 7906505). In another embodiment, the BCL-2 anti-apoptotic protein inhibitor is a small molecule macrocyclic compound (see, e.g., international patent application publication No. WO 2006/127364, U.S. patent No. 7777076). In yet another embodiment, the BCL-2 anti-apoptotic protein inhibitor is an isoxazolidine compound (see, e.g., international patent application publication No. WO 2008/060569, U.S. patent No. 7851637, U.S. patent No. 7842815).

In certain embodiments, the senolytic agent is a compound that is an inhibitor of Bcl-2, Bcl-w, and Bcl-xL, such as ABT-263 or ABT-737. In certain particular embodiments, the senolytic agent is a compound as shown below, or a pharmaceutically acceptable salt, stereoisomer, tautomer, or prodrug thereof, that depicts the structure of ABT-263. ABT-263 is also known in the art as Navitoclax.

Akt kinase inhibitors

In certain embodiments, the senolytic agent is an Akt kinase inhibitor. For example, senolytic scavengers can be small molecule compounds that inhibit Akt and analogs thereof. In some embodiments, the senolytic agent is a compound that selectively inhibits Akt1, Akt2, and Akt3 relative to other protein kinases.

Akt inhibitors (which may also be referred to as Akt kinase inhibitors or AKT kinase inhibitors) can be divided into six broad classes based on their mechanism of action (see, e.g., Bhutani et al, Infectious Agents and Cancer 2013,8:49doi: 10.1186/1750-. Akt is also known in the art as protein kinase b (pkb). The first class includes ATP competitive inhibitors of Akt and includes compounds that inhibit Akt2 and Akt1 such as CCT128930 and GDC-0068. This class also includes pan-Akt kinase inhibitors such as GSK2110183 (afurertib), GSK690693 and AT 7867. The second class includes lipid-based inhibitors of Akt that act by inhibiting the production of PIP3 by PI 3K. Phosphatidylinositol analogs such as Calbiochem Akt inhibitors I, II and III or other PI3K inhibitors such as PX-866 use this mechanism. This category also includes compounds such as piperacillin (KRX-0401) (Aeterna Zentaris/Keryx). The third class includes a group of compounds known as false substrate inhibitors. They include compounds such as AKTide-2T and FOXO3 hybrids. The fourth class consists of allosteric inhibitors of the AKT kinase domain and includes compounds such as MK-2206(8- [4- (1-aminocyclobutyl) phenyl ] -9-phenyl-2H- [1,2,4] triazolo [3,4-f ] [1,6] naphthyridin-3-one; dihydrochloride) (Merck & Co.) (see, e.g., U.S. Pat. No. 7576209). The fifth class consists of antibodies and includes molecules such as GST-anti-Akt 1-MTS. The last category includes compounds that interact with the PH domain of Akt, and includes triciribine and PX-316. Other compounds described in the art that act as AKT inhibitors include, for example, GSK-2141795(GlaxoSmithKline), VQD-002, miltefosine, AZD5363, GDC-0068, and API-1. Techniques for determining the activity of AKT inhibitors are routinely performed by those skilled in the art.

In a particular embodiment, the senolytic agent is a compound that is an inhibitor of Akt kinase, which has the structure shown below (also referred to herein and in the art as MK-2206), 8- [4- (1-aminocyclobutyl) phenyl ] -9-phenyl-2H- [1,2,4] triazolo [3,4-f ] [1,6] naphthyridin-3-one), or a pharmaceutically acceptable salt, stereoisomer, tautomer, or prodrug thereof. Dihydrochloride salts are shown.

In certain embodiments, at least one senolytic agent can be administered with at least one other senolytic agent, the two or more senolytic agents acting additively or synergistically to selectively kill senescent cells. In particular embodiments, methods of using senolytic agents are provided, wherein the senolytic agent alters a cell survival signaling pathway or an inflammatory pathway, or both a cell survival signaling pathway and an inflammatory pathway, in a senescent cell. In other particular embodiments, the methods comprise the use of at least two senolytic agents, wherein at least one senolytic agent and the second senolytic agent are each different and independently alter one or both of a survival signaling pathway and an inflammatory pathway in the senescent cell. For convenience, when two or more senolytic agents are described herein as being used in combination, one senolytic agent will be referred to as a first senolytic agent, another senolytic agent will be referred to as a second senolytic agent, and so on. In certain other embodiments, the methods described herein comprise administering at least three senolytic agents (a first senolytic agent, a second senolytic agent, and a third senolytic agent). The adjectives first, second, third, etc., are used herein for convenience only and should not be construed as describing an order of administration, preference, or level of senolytic activity or other parameter unless expressly described otherwise. In particular embodiments, when two or more senolytic agents are used in the methods described herein, each senolytic agent is a small molecule. In certain other embodiments, the methods described herein comprise administering at least three senolytic agents (a first senolytic agent, a second senolytic agent, and a third senolytic agent). In certain embodiments, the use of at least two senolytic agents results in killing a significant increase in senescent cells as compared to the use of each senolytic agent alone. In other particular embodiments, the use of at least two senolytic agents results in a significant killing of senescent cells compared to the use of each senolytic agent alone, and the effects may be additive or synergistic. In certain embodiments, the at least two senolytic agents are each different and are selected from (1) an inhibitor of one or more BCL-2 anti-apoptotic protein family members, wherein the inhibitor inhibits at least BCL-xL; (e.g., Bcl-2/Bcl-xL/Bcl-w inhibitors; Bcl-2/Bcl-xL inhibitors, selective Bcl-xL inhibitors, or Bcl-xL/Bcl-w inhibitors); inhibitors specific for Akt kinase; an MDM2 inhibitor. In a particular embodiment, the second senolytic agent is administered when the at least one senolytic agent administered to the subject in need thereof is an inhibitor of one or more BCL-2 anti-apoptotic protein family members, wherein the inhibitor at least inhibits BCL-XL (e.g., a BCL-2/BCL-XL/BCL-w inhibitor, a BCL-2/BCL-XL inhibitor, a selective BCL-XL inhibitor, or a BCL-XL/BCL-w inhibitor). In other certain embodiments, one of the two senolytic agents is an inhibitor of one or more BCL-2 anti-apoptotic protein family members, wherein the inhibitor inhibits at least BCL-xL, and the second senolytic agent is an MDM2 inhibitor. In a more specific embodiment, the second senolytic agent is administered when the at least one senolytic agent administered to the subject in need thereof is a selective Bcl-xL inhibitor. In a more specific embodiment, when the at least one senolytic agent administered to the subject in need thereof is an MDM2 inhibitor, a second senolytic agent is administered. In a more specific embodiment, when the at least one senolytic agent administered to a subject in need thereof is an Akt kinase inhibitor, a second senolytic agent is administered. In even more particular embodiments, an inhibitor of one or more BCL-2 anti-apoptotic protein family members, wherein the inhibitor inhibits at least BCL-xL, is used alone or in combination with another senolytic agent that also acts as an inhibitor of one or more BCL-2 anti-apoptotic protein family members, wherein the inhibitor inhibits at least BCL-xL or a different senolytic agent as described herein. In certain embodiments, one or more inhibitors of BCL-2 anti-apoptotic protein family members, wherein the inhibitor inhibits at least Bcl-xL, is combined with an inhibitor of Akt kinase. By way of non-limiting example, the Bcl-2/Bcl-xL/Bcl-w inhibitor ABT-263 can be used in combination with an Akt kinase inhibitor (e.g., MK 2206).

In other particular embodiments, in the methods for treating a senescence-associated disease or disorder, the MDM2 inhibitor as a senolytic agent is used in combination with at least one other senolytic agent; the other senolytic agent (which may be referred to as a second senolytic agent for convenience) may be another MDM2 inhibitor or may be a senolytic agent that is not a MDM2 inhibitor. In one embodiment, an inhibitor that inhibits at least a Bcl-2 anti-apoptotic family member of BCL-xL is used in combination with an AKT inhibitor. In a more specific embodiment, the inhibitor of the Bcl-2 anti-apoptotic family member is ABT-263, ABT-737, or WEHI-539, and the AKT inhibitor is MK-2206.

In certain other embodiments, the methods described herein comprise administering at least three senolytic agents (a first senolytic agent, a second senolytic agent, and a third senolytic agent).

mTOR, nfkb and PI3-k pathway inhibitors: small molecule compounds that may be used with the senolytic agents described herein in a method for selectively killing senescent cells and treating a senescence-associated disease or disorder may be small molecule compounds that inhibit one or more of the mTOR, nfkb, and PI3-k pathways. Also provided are methods for selectively killing senescent cells and treating a senescence-associated disease or disorder, as described herein, wherein the methods comprise administering at least one senolytic agent to a subject in need thereof, which methods may further comprise administering an inhibitor of one or more of the mTOR, nfkb, and PI3-k pathways. Inhibitors of these pathways are known in the art.

Examples of mTOR inhibitors include sirolimus, temsirolimus (temsirolimus), everolimus, ridaforolimus, 32-deoxyrapamycin, zotarolimus, PP242, INK128, PP30, Torin1, Ku-0063794, WAY-600, WYE-687, and WYE-354. Inhibitors of the NF-. kappa.B pathway include, for example, TPCA-1 (an IKK2 inhibitor) which abolishes NF-. kappa.B activity; BAY 11-7082(IKK inhibitors, less selective for IKK1 and IKK 2); and MLN4924 (NEDD8 activating enzyme (NAE) -inhibitors); and an MG 132.

Examples of PI3-k inhibitors that may also inhibit the mTOR or AKT pathway include perifosine (KRX-0401), idelalisib, PX-866, IPI-145, BAY 80-6946, BEZ235, RP6530, TGR 1201, SF1126, INK1117, GDC-0941, BKM120, XL147 (SAR 24576 408), XL765(SAR 24409), Palomid 529, GSK1059615, GSK 066993, ZSTK474, PWT33597, IC87114, TG100-115, CAL263, RP6503, PI-103, GNE-477, DC-907, AEZS-136, BYL719, BKM120, GDC-0980, GDC-0032, and MK 2206.

Small molecule compounds-salts and general synthetic procedures. Small molecule compounds described herein as senolytic agents include physiologically acceptable salts (i.e., pharmaceutically acceptable salts), hydrates, solvates, polymorphs, metabolites, and prodrugs of senolytic agents. Further information on metabolism can be obtained from The Pharmacological Basis of Therapeutics, 9 th edition, McGraw-Hill (1996). Metabolites of the compounds disclosed herein can be identified by administering the compound to a host and analyzing a tissue sample from the host, or by incubating the compound with hepatocytes in vitro and analyzing the resulting compound. Both of these methods are well known in the art.

The compounds described herein can generally be used as the free acid or as the free base. Alternatively, the compounds may be used in the form of acid or base addition salts. Acid addition salts of the free base amino compounds can be prepared according to methods well known in the art and can be formed from organic and inorganic acids. Suitable organic acids include, but are not limited to, maleic acid, fumaric acid, benzoic acid, ascorbic acid, succinic acid, methanesulfonic acid, acetic acid, oxalic acid, propionic acid, tartaric acid, salicylic acid, citric acid, gluconic acid, lactic acid, mandelic acid, cinnamic acid, aspartic acid, stearic acid, palmitic acid, glycolic acid, glutamic acid, malonic acid, and benzenesulfonic acid. Suitable inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and nitric acid. Base addition salts of the free acid compounds of the compounds described herein can also be prepared according to methods well known in the art and can be formed from organic and inorganic bases. Additional salts include those salts in which the counterion is a cation. Suitable inorganic bases include, but are not limited to, hydroxides or other salts of sodium, potassium, lithium, ammonium, calcium, barium, magnesium, iron, zinc, copper, manganese, aluminum, and the like, and organic bases such as substituted ammonium salts (e.g., dibenzylammonium, benzylammonium, 2-hydroxyethylammonium). Other salts include those in which the counterion is an anion, such as adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, formates, fumarates, glucoheptonates, glycerophosphates, gluconates, hemisulfates, heptanoates, hexanoates, hydroiodiates, 2-hydroxy-ethanesulfonates, lactobionates, lactates, laurylsulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamonates, pectinates, persulfates, laurylsulfates, malates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamonates, pectinates, persulfates, persulfa, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate. Thus, the term "pharmaceutically acceptable salt" of a compound as described herein is intended to encompass any and all pharmaceutically suitable salt forms.

Compounds may sometimes be described as anionic species. One of ordinary skill in the art will recognize that the compounds are present in an equimolar ratio of the cations. For example, the compounds described herein may be present in fully protonated form, or as salts such as sodium, potassium, ammonium, or in combination with any of the inorganic bases described above. When more than one anionic species is described, each anionic species may be present independently as a protonated species or as a salt species. In some particular embodiments, the compounds described herein are present as sodium salts. In other particular embodiments, the compounds described herein are present as potassium salts.

In addition, some crystalline forms of any of the compounds described herein may exist as polymorphs and as such are included and contemplated by the present disclosure. In addition, some compounds may form solvates with water or other organic solvents. Crystallization typically produces solvates of the disclosed compounds. As used herein, the term "solvate" refers to an aggregate of one or more molecules comprising any of the disclosed compounds and one or more solvent molecules. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds disclosed herein may exist as hydrates including monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate and the like, as well as the corresponding solvated forms. Certain embodiments of the compounds may be true solvates, while in other cases, some embodiments of the compounds may retain only extraneous water or only a mixture of water plus some extraneous solvent.

In general, the compounds used in the methods described herein can be made starting from commercially available chemicals and/or from compounds described in the chemical literature according to organic synthesis techniques known to those skilled in the art. Specific and similar reactants can also be identified by known Chemical indexes made by the Chemical abstracts service of the American Chemical Society, available in most libraries of public and university, and by online-databases, which may be linked to the American Chemical Society, Washington, d.c. for more details. Known but not commercially available chemicals in the catalog can be prepared by a custom chemical synthesis room, where many standard chemical supply rooms (e.g., those listed above) provide custom synthesis services. References to the preparation and selection of pharmaceutically acceptable Salts of the present disclosure are p.h.stahl & c.g.wermuth "Handbook of Pharmaceutical Salts," Verlag Helvetica Chimica Acta, Zurich, 2002. Methods known to those of ordinary skill in the art can be identified by various reference books and databases. Suitable references and articles detail the synthesis of reactants useful in the preparation of the compounds described herein or provide references to articles describing the preparation.

Assays and techniques for identifying senolytic agents are described in more detail herein. In addition, one skilled in the art of pharmaceutical chemistry can also consider other properties of small molecules, such as solubility, bioavailability, pharmacokinetics, Lipinski penta-regulation, and the like, in order to identify and select small compounds as senolytic agents.

Polypeptides, antibodies and nucleic acids

In certain other embodiments, the senolytic agent can be a polypeptide, a peptide, an antibody, an antigen-binding fragment (i.e., peptides and polypeptides comprising at least one Complementarity Determining Region (CDR)), a peptibody, a recombinant viral vector, or a nucleic acid. In certain embodiments, the senolytic agent is an antisense oligonucleotide, siRNA, shRNA, or peptide. For example, senolytic agents such as polypeptides, antibodies, nucleic acids, and the like include, for example, an MDM2 inhibitor, a BCL-2 family inhibitor, or an Akt kinase inhibitor. In other embodiments, polypeptides, peptides, antibodies (including antigen-binding fragments thereof) that specifically bind to a ligand or target protein of a small molecule senolytic agent described herein can be used in assays and methods for characterizing or monitoring the use of the small molecule senolytic agent.

Polynucleotides or oligonucleotides that specifically hybridize to a portion of the mRNA encoding a target protein (e.g., Bcl-xL, Bcl-2, Bcl-w, MDM2, Akt) of a cell (which is a senescent cell or a cell in a disease microenvironment) can induce senescence in the cell by aging, biological injury (i.e., cellular injury) drug therapy, or environmental stimuli. In other embodiments, the target protein may be a ligand or protein downstream or upstream of a cell survival pathway or an inflammation pathway or an apoptosis pathway. Polynucleotides and oligonucleotides can be complementary to at least a portion of a nucleotide sequence encoding a target polypeptide (e.g., short interfering nucleic acids, antisense polynucleotides, ribozymes, or peptide nucleic acids), and can be used to alter gene and/or protein expression. Such polynucleotides that specifically bind or hybridize to a nucleic acid molecule encoding a target polypeptide can be prepared using nucleotide sequences available in the art. In another embodiment, nucleic acid molecules such as non-sequence specific aptamers may also be used to alter gene and/or protein expression.

Antisense polynucleotides bind to nucleic acids such as mRNA or DNA in a sequence specific manner. Identification of oligonucleotides and ribozymes for use as antisense agents and identification of DNA encoding a target gene for targeted delivery include methods well known in the art. For example, the desired properties, length and other characteristics of such oligonucleotides are well known. Antisense technology can be used to control gene expression by interfering with the binding of polymerases, transcription factors, or other regulatory molecules (see, e.g., Gee et al, In Huber and pcr, Molecular and immunological applications, Futura Publishing co. (mt. kisco, NY; 1994)).

Short interfering RNAs can be used to modulate (reduce or inhibit) expression of a gene encoding a target polypeptide of interest (see, e.g., the examples herein). Small nucleic acid molecules such as short interfering rna (sirna), micro rna (mirna), and short hairpin rna (shrna) molecules can be used to modulate the expression of a target protein according to the methods described herein. siRNA polynucleotides preferably comprise double-stranded RNA (dsRNA), but may comprise single-stranded RNA (see, e.g., Martinez et al, Cell 110:563-74 (2002)). The siRNA polynucleotide may comprise other naturally occurring, recombinant or synthetic single-or double-stranded nucleotide polymers (ribonucleotides or deoxyribonucleotides or a combination of both) and/or nucleotide analogs as provided herein and known and used by those of skill in the art.

Unless otherwise indicated, the term "siRNA" refers to double-stranded interfering RNA. Typically, the siRNA is a double-stranded nucleic acid molecule comprising two strands of nucleotides, each strand having from about 19 to about 28 nucleotides (i.e., about 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). In certain embodiments, each strand is 19, 20, 21, 22, or 23 nucleotides. In other specific embodiments, the siRNA comprises two strands of nucleotides, each strand having about 15, 16, 17, or 18 nucleotides. In certain other embodiments, one strand of a double stranded siRNA is at least two nucleotides longer, e.g., one strand may have a two base overhang (e.g., TT) at one end (typically the 3' end).

Short hairpin interfering RNA molecules comprise a sense strand and an antisense strand of a stem-loop or hairpin structured interfering RNA (e.g., shRNA). The shRNA can be expressed from a DNA vector in which a DNA oligonucleotide encoding a sense interfering RNA strand is linked to a DNA oligonucleotide encoding a reverse complementary antisense interfering RNA strand by a short spacer. If desired, a 3' terminal T and a nucleotide forming a restriction site may be added. The resulting RNA transcript folds back on itself to form a stem-loop structure.

In addition to siRNA molecules, other interfering RNA and RNA-like molecules may also interact with RISC and silenced gene expression, such as short hairpin RNA (shrna), single stranded siRNA, microrna (mirna), and dicer-substrate 27-mer duplexes. Such RNA-like molecules may contain one or more chemically modified nucleotides, one or more non-nucleotides, one or more deoxyribonucleotides, and/or one or more non-phosphodiester linkages. RNA or RNA-like molecules that can interact with RISC in gene expression and participate in RISC-related changes may be referred to herein as "interfering RNA" or "interfering RAN molecules". In some cases, single-stranded interfering RNA causes mRNA silencing, but less efficiently than double-stranded RNA.

One skilled in the art will also recognize that RNA molecules such as siRNA, miRNA, shRNA can be chemically modified to confer increased stability against nuclease degradation while retaining the ability to bind to target nucleic acids that may be present in the cell. The RNA can be modified at any position of the molecule as long as the modified RNA binds to the target sequence of interest and is resistant to enzymatic degradation. Modifications of the siRNA can be in the nucleotide base, ribose, or phosphate. For example, the 2' position of the ribose can be modified using any of a number of different methods commonly practiced in the art. RNA can be chemically modified by the addition of a halo group such as fluoro. Other chemical moieties that have been used to modify RNA molecules include methyl, methoxyethyl, and propyl groups (see, e.g., U.S. patent No. 8,675,704).

In particular embodiments, the polynucleotide or oligonucleotide (e.g., including shRNA) may be delivered by a recombinant vector that already incorporates the polynucleotide or oligonucleotide of interest. In other embodiments, the recombinant viral vector may be a recombinant expression vector into which is inserted a polynucleotide sequence encoding an antibody, antigen-binding fragment, polypeptide, or peptide that inhibits a protein in a cell survival pathway or an inflammatory pathway, including proteins described herein such as Bcl-xL, Bcl-2, Bcl-w, MDM2, and Akt, such that the coding sequence is operably linked to one or more regulatory control sequences to drive expression of the polypeptide, antibody, antigen-binding fragment, or peptide. The recombinant vector or recombinant expression vector may be a viral recombinant vector or a viral recombinant expression vector. Exemplary viral vectors include, without limitation, lentiviral vector genomes, poxviral vector genomes, vaccinia viral vector genomes, adenoviral vector genomes, adeno-associated viral vector genomes, herpes viral vector genomes, and alphaviral vector genomes. Viral vectors may be live, attenuated, conditionally replicating or replication defective, and are typically nonpathogenic (defective), replicable viral vectors. Procedures and techniques for designing and generating such viral vectors are well known to those skilled in the art and are commonly practiced.

In certain particular embodiments, the senolytic agent that can be used in the methods described herein is an antisense oligonucleotide. By way of non-limiting example, the previously described BCL-xL specific antisense oligonucleotides can be used in the methods described herein (see, e.g., PCT publication WO 00/66724; Xu et al, Intl.J.cancer 94:268-74 (2001); Olie et al, J. invest.Dermatol.118: 505-.

In certain embodiments, the senolytic agent useful in the methods described herein is a peptide. For example and in certain embodiments, the BCL-xL selective peptide inhibitor is a BH3 peptide mimetic. Examples of BCL-xL selective BH3 peptide mimetics include those previously described (see, e.g., Kutzki et al, j.am.chem.soc.124:11838-39 (2002); Yin et al, bioorg. med.chem.lett.22:1375-79 (2004); Matsumura et al, faeb J.7:2201 (2010)).

In certain embodiments, senolytic agents useful in the methods described herein do not include a polynucleotide encoding an exonuclease, EXO1, or a vector comprising a polynucleotide encoding an EXO1 enzyme (including viral vectors), or a fragment thereof (i.e., a polynucleotide encoding an EXO1 enzyme, a fragment of such a polynucleotide, or a vector comprising such a polynucleotide is excluded). Senescence eliminators useful in the methods described herein also do not include EXO1 enzyme polypeptides (i.e., EXO1 enzyme is excluded) or biologically active peptides or polypeptide fragments thereof. Furthermore, such molecules are not inhibitors of cell signaling pathways such as one or both of inflammatory pathways or cell survival pathways; in contrast, EXO1 encodes a 5 '-3' exonuclease that degrades capped defective telomeres (see, e.g., international patent application No. WO 2006/018632).

The senolytic agent described herein can be a polypeptide that is an antibody or antigen-binding fragment. The antigen-binding fragment may be F (ab')₂Fab, Fab', Fv, and Fd, and also includes peptides or polypeptides comprising at least one Complementarity Determining Region (CDR). The antibody may be an internalizing antibody or antigen-binding fragment that is internalized by the senescent cell via interaction with a target protein.

The binding characteristics of an antibody to its cognate antigen can generally be determined and evaluated using methods readily performed by those of ordinary skill in the art (see, e.g., Harlow et al, Antibodies: A Laboratory Manual, Cold spring Harbor Laboratory (1988)). As used herein, an antibody is considered "immunospecific for", "specific for" or "specifically binds" to an antigen if the antibody reacts with the polypeptide at detectable levels. Conventional techniques such as those described by Scatchard et al (Ann.N.Y.Acad.Sci.USA 51:660(1949)) and by surface plasmon resonance (SPR; BIAcore)^TMBiosensor, Piscataway, NJ) readily determined the affinity of an antibody for its antigen binding fragment.

The antibody may be polyclonal or monoclonal. The variable region or one or more Complementarity Determining Regions (CDRs) can be identified and isolated from an antigen binding fragment or peptide library. The antibody or antigen binding fragment may be recombinantly engineered and/or recombinantly produced. The antibody may belong to any immunoglobulin class, such as IgG, IgE, IgM, IgD, or IgA, and may be obtained or derived from animals, such as fowl (e.g., chickens) and mammals, including but not limited to mice, rats, hamsters, rabbits or other rodents, cows, horses, sheep, goats, camels, humans, or other primates. For use in human subjects, antibodies and antigen-binding fragments are typically human, humanized or chimeric to reduce the immunogenic response of the subject to non-human peptide and polypeptide sequences.

The antibody can be a monoclonal antibody that is a human antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, or an antigen-binding fragment (e.g., F (ab')₂Fab, Fab', Fv and Fd). The antigen-binding fragment can also be any synthetic or genetically engineered protein (see, e.g., Hayden et al, Curropin. Immunol. 9:201-12 (1997); Coloma et al, nat. Biotechnol.15:159-63 (1997); U.S. Pat. No. 5,910573); holliger et al, Cancer Immunol.Immunother.45: 128-30 (1997); drakeman et al, Expert opin, investig. drugs 6:1169-78 (1997); koelemij et al, J.Immunother.22:514-24 (1999); marvin et al, Acta Pharmacol.sin.26:649-58 (2005); das et al, Methods mol. Med.109:329-46 (2005); international patent application Nos. PCT/US91/08694 and PCT/US91/04666), and from phage display peptide libraries (see, e.g., Scott et al, Science 249:386 (1990); devlin et al, Science 249:404 (1990); cwirla et al, Science 276:1696-99 (1997); U.S. Pat. nos. 5,223,409; U.S. patent nos. 5,733,731; U.S. Pat. nos. 5,498,530; U.S. Pat. nos. 5,432,018; U.S. Pat. nos. 5,338,665; 1994; U.S. patent nos. 5,922,545; international application publication nos. WO 96/40987 and WO 98/15833). Peptides that are the smallest recognition unit or CDR (i.e., any one or more of the three CDRs present in the heavy chain variable region and/or one or more of the three CDRs present in the light chain variable region) can be identified by computer modeling techniques that can be used to compare and predict peptide sequences that will specifically bind to a target protein of interest (see, e.g., Bradley et al, Science 309:1868 (2005); Schueler-Furman et al, Science 310:638 (2005)). Useful strategies for designing humanized antibodies are described in the art (see, e.g., Jones et al, Nature 321:522-25 (1986); Riechmann et al, Nature 332:323-27 (1988); padlan et al, FASEB9:133-39 (1995); chothia et al, Nature, 342:377-83 (1989)).

Senescent cells

The senolytic agents described herein can be used to selectively kill or destroy senescent cells in a clinically significant or biologically significant manner. As discussed in detail herein, the one or more senescent scavengers are used in an amount and for a time sufficient to selectively kill established senescent cells, but insufficient to kill non-senescent cells (destroy non-senescent cells, cause death of non-senescent cells) in a clinically significant or biologically significant manner. The senolytic agent can selectively kill one or more types of senescent cells (e.g., senescent preadipocytes, senescent endothelial cells, senescent fibroblasts, senescent neurons, senescent epithelial cells, senescent mesenchymal cells, senescent smooth muscle cells, senescent macrophages, or senescent chondrocytes).

Senescent cells can exhibit any one or more of the following seven characteristics. (1) The senescence growth arrest is essentially permanent and cannot be reversed by known physiological stimuli. (2) Senescent cells increase in size, sometimes by more than a factor of two relative to the size of non-senescent counterparts. (3) Senescent cells express senescence-associated beta-galactosidase (SA-beta-gal) which reflects, in part, increased lysosomal mass. (4) most senescent cells express p 16. sup. INK4a, and p 16. sup. INK4a is not normally expressed by quiescent or terminally differentiated cells. (5) Senescent cells with persistent DDR signaling carry persistent nuclear foci, known as DNA segments with chromatin changes that potentiate senescence (DNA-SCARS). These foci contain activated DDR proteins and are distinguishable from transient lesion foci. DNA-SCARS include dysfunctional telomeres or telomere dysfunction-induced foci (TIF). (6) Senescent cells express and can secrete senescence-associated molecules, which in some cases can be observed in the presence of persistent DDR signaling, the expression of which in some cases may be dependent on persistent DDR signaling. (7) The nucleus of senescent cells loses structural proteins such as lamin B1 or chromatin-associated proteins such as histones and HMGB 1. See, e.g., Freund et al, mol. biol. cell 23:2066-75 (2012); davalos et al, J.cell biol. 201:613-29 (2013); ivanov et al, J.cell biol.DOI 10.1083/jcb.201212110, pages 1-15; published online in 2013, 7 months and 1 day; funayama et al, J.cell biol.175:869-80 (2006)).

Senescent cells and senescent cell-associated molecules can be detected by techniques and procedures described in the art. For example, tissues can be analyzed for the presence of senescent cells by histochemical or immunohistochemical techniques that detect the senescence marker SA- β galactosidase (SA- β gal) (see, e.g., Dimri et al, Proc. Natl. Acad. Sci. USA92: 9363-. The presence of the senescent cell-associated polypeptide p16 can be determined by any of a variety of immunochemical methods practiced in the art, such as immunoblot analysis. Expression of p16mRNA in a cell can be measured by a variety of techniques practiced in the art, including quantitative PCR. The presence and levels of senescent cell-associated polypeptides (e.g., a polypeptide of SASP) can be determined by using an automated high-throughput assay, such as an automated Luminex array assay described in the art (see, e.g., Coppe et al, PLoS Biol 6:2853-68 (2008)).

The presence of senescent cells can also be determined by detecting senescent cell-associated molecules, including growth factors, proteases, cytokines (e.g., inflammatory cytokines), chemokines, cell-associated metabolites, reactive oxygen species (e.g., H)₂O₂) And other molecules that stimulate inflammation and/or other biological effects or responses that may promote or exacerbate the underlying disease in the subject. Senescence cell-associated molecules include those described in the art as comprising a senescence-associated secretory phenotype (SASP, i.e., it includes secreted factors that can make up the pro-inflammatory phenotype of senescent cells), a secretory proteome that conveys senescence information, and a DNA Damage Secretory Program (DDSP). As described in the art, these groupings of senescent cell-associated molecules contain a common molecule and are not intended to describe three separate distinct groupings of molecules. Senescence cell-associated molecules include certain expressed and secreted growth factors, proteases, cytokines and proteins that may have potent autocrine and paracrine activityOther factors (see, e.g., Coppe et al, supra; Coppe et al, J.biol. chem.281:29568-74 (2006); Coppe et al, PLoSONE5:39188 (2010); Krtolica et al, Proc. Natl. Acad. Sci.U.S.A.98:12072-77 (2001); Parriello et al, J.cell Sci.118: 485-96 (2005)). ECM-related factors include inflammatory proteins and mediators of ECM remodeling, and they are strongly induced in senescent cells (see, e.g., Kuilman et al, Nature Reviews9:81-94 (2009)). Other senescence cell-associated molecules include extracellular polypeptides (proteins) that are collectively described as the DNA Damage Secretion Program (DDSP) (see, e.g., Sun et al, Nature Medicine 18: 1359-. Senescent cell-associated proteins also include cell surface proteins (or receptors) expressed on senescent cells, including proteins that are present or absent on the cell surface of non-senescent cells in detectably lower amounts.

Senescent cell-associated molecules include secreted factors (e.g., SASPs) that can make up the pro-inflammatory phenotype of senescent cells. These factors include, but are not limited to, GM-CSF, GRO α, β, γ, IGFBP-7, IL-1 α, IL-6, IL-7, IL-8, MCP-1, MCP-2, MIP-1 α, MMP-1, MMP-10, MMP-3, amphiregulin, ENA-78, eotaxin-3, GCP-2, GITR, HGF, ICAM-1, IGFBP-2, IGFBP-4, IGFBP-5, IGFBP-6, IL-13, IL-1 β, MCP-4, MIF, MIP-3 α, MMP-12, MMP-13, MMP-14, NAP2, oncostatin M, osteoprotegerin, PIGF, RANTES, sgp130, TIMP-2, TRAIL-R3, Acrp30, angiogenin, FGF, BLC, bFGF, BLC, BTC, CTACK, EGF-R, Fas, FGF-7, G-CSF, GDNF, HCC-4, I-309, IFN- γ, IGFBP-1, IGFBP-3, IL-1R1, IL-11, IL-15, IL-2R- α, IL-6R, I-TAC, leptin, LIF, MMP-2, MSP-a, PAI-1, PAI-2, PDGF-BB, SCF, SDF-1, sTNF RI, sTNF RII, thrombopoietin, TIMP-1, tPA, uPA, uRI, VEGF, MCP-3, IGF-1, TGF- β 3, MIP-1-delta, IL-4, FGF-7, FGF-BB, IL-16, PDGF-4, PAR, MCP-4, IL-10, TIMP-1, Fit-3 ligand, PDG-3, PAR-1, PDGF-1, and PAR, ICAM-1, Axl, CNTF, INF-gamma, EGF, BMP-6. Additional identified factors, including those sometimes referred to in the art as secretory group (SMS) factors that convey senescence information, some of which are included in the list of SASP polypeptides, include, without limitation, IGF1, IGF2 and IGF2R, IGFBP3, IDFBP5, IGFBP7, PAl1, TGF- β, WNT2, IL-1 α, IL-6, IL-8 and CXCR2 binding chemokines. Cell-related molecules also include, without limitation, the factors described in Sun et al, Nature Medicine (supra), and include, for example, the products of the genes MMP, WNT16, SFRP, MMP, SPINK, MMP, ENPP, EREG, BMP, ANGPTL, AREG, ANGPT, CCK, THBD, CXCL, NOV, GAL, NPPC, FAM150, CST, GDNF, MUCL, NPTX, TMEM155, EDN, PSG, ADAMTS, CD, PPBP, CXCL, MMP, CST, PSG, PCOLE, PSG, TNFSF, C17orf, CA, FGF, IL, BMP, MATN, TFP, SERPINI 1, TNFRSF, and IL 23. Senescent cell-associated proteins also include cell surface proteins (or receptors) expressed on senescent cells, including proteins that are present or absent on the cell surface of non-senescent cells in detectably lower amounts.

In certain embodiments, a senolytic agent that at least selectively kills senescent preadipocytes can be used to treat diabetes (particularly type 2 diabetes), metabolic syndrome, or obesity. In other embodiments, the senolytic agent is at least capable of selectively killing senescent endothelial cells, senescent smooth muscle cells, and/or senescent macrophages. Such senolytic agents are useful for treating cardiovascular disease (e.g., atherosclerosis). In other particular embodiments, the senolytic agent is capable of at least selectively killing senescent fibroblasts. In another embodiment, the senolytic agent can at least selectively kill senescent neurons, including dopamine-producing neurons. In another embodiment, the senolytic agent can kill at least senescent retinal pigment epithelial cells or other senescent epithelial cells (e.g., senescent epithelial cells of the lung or senescent kidney (kidney) epithelial cells). At least selectively killing senescent lung epithelial cells can be used to treat lung diseases such as chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis. In other embodiments, the senolytic agent can at least selectively kill senescent immune cells (e.g., senescent macrophages). In another embodiment, the senolytic agent can kill at least senescent chondrocytes, which can be used to treat an inflammatory disorder such as osteoarthritis.

Methods for selectively killing senescent cells

Provided herein are methods for selectively killing senescent cells to treat or prevent (reduce the likelihood of) a senescence-associated disease or disorder, and comprising the use of a senolytic agent as described herein. As described herein, these senolytic agents are administered in a manner that is considered ineffective for treating cancer. Because the methods of treating senescence-associated diseases with the senolytic agents described herein comprise one or more of reducing the daily dose, reducing the cumulative dose within a single treatment cycle, or reducing the cumulative dose of the senolytic agent (e.g., an MDM2 inhibitor; an inhibitor that inhibits at least one Bcl-2 anti-apoptotic family member of a Bcl-xL; an Akt inhibitor) over multiple treatment cycles as compared to the amount required for cancer treatment, the likelihood of the occurrence of one or more adverse reactions (i.e., side effects) associated with treating the subject according to a regimen optimized for treating cancer is reduced.

A treatment regimen for the method of treating a senescence-associated disease or disorder comprises administering a senolytic agent for a time and in an amount sufficient to selectively kill senescent cells. In certain embodiments, the senolytic agent is administered within a treatment cycle comprising a treatment course followed by a non-treatment interval. A course of treatment administered herein refers to a limited time frame over which one or more doses of senolytic agent are administered for one or more days. This limited time frame may also be referred to herein as a therapeutic window.

In one embodiment, provided herein is a method for treating a non-cancer, senescence-associated disease or disorder, comprising administering to a subject in need thereof a small molecule senolytic agent that selectively kills senescent cells and is administered within a treatment cycle. In particular embodiments, the method comprises administering the senolytic agent for at least two treatment cycles. In particular embodiments, the non-treatment interval may be at least about 2 weeks or at least about 0.5-12 months, such as at least about one month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months (i.e., 1 year). In other certain specific embodiments, the non-treatment interval is 1-2 years or 1-3 years or longer. In certain embodiments, each treatment course is no longer than about 1 month, no longer than about 2 months, or no longer than about 3 months; or no longer than 1,2, 3,4,5,6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days.

In certain embodiments, the treatment window (i.e., treatment course) is only one day. In certain other embodiments, a single course of treatment occurs in no more than 2,3, 4,5,6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. During such a therapeutic window, the senolytic agent can be administered for at least two days (i.e., two days or more), wherein there are variable days of non-administration of the agent between the at least two days of administration. In other words, within a treatment session of two or more days of administration of the senolytic agent, the treatment session may have one or more intervals of one or more days of non-administration of the senolytic agent. By way of non-limiting example, when the senolytic agent is administered for 2 or more days during a treatment course of no more than 21 days, the agent can be administered for any total number of days of 2,3, 4,5,6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. In certain embodiments, the senolytic agent is administered to the subject during a treatment course of 3 days or more, and the agent can be administered every two days (i.e., every other day). In certain other embodiments, the senolytic agent can be administered every three days (i.e., every two days) when the subject is administered the senolytic agent for a treatment window of 4 days or more. In one embodiment, the senolytic agent is administered for at least two days (i.e., 2 days or more) during a treatment course of at least 2 days and not more than about 21 days (i.e., about 2-21 days), at least 2 days and not longer than about 14 days (i.e., about 2-14 days), at least 2 days and not longer than about 10 days (i.e., about 2-10 days), or at least 2 days and not longer than about 9 days (i.e., about 2-9 days), or at least 2 days and not longer than about 8 days (i.e., about 2-8 days). In other particular embodiments, the senolytic agent is administered for at least two days (i.e., 2 days or more) during a treatment window of at least 2 days and not longer than about 7 days (i.e., about 2-7 days), at least 2 days and not longer than about 6 days (i.e., about 2-6 days), or at least 2 days and not more than about 5 days (i.e., about 2-5 days), or at least 2 days and not longer than about 4 days (i.e., about 2-4 days). In yet another embodiment, the treatment window is at least 2 days and not longer than 3 days (i.e., 2-3 days) or 2 days. In certain specific embodiments, the treatment course is no longer than 3 days. In other embodiments, the treatment course is no longer than 5 days. In other particular embodiments, the course of treatment is no longer than 7 days, 10 days, or 14 days or 21 days. In certain embodiments, the senolytic agent is administered for at least two days (i.e., 2 days or more) during a treatment window of at least 2 days and not longer than about 11 days (i.e., 2-11 days); or administering the senolytic agent for at least two days (i.e., 2 days or more) during a treatment window of at least 2 days and not longer than about 12 days (i.e., 2-12 days); or administering the senolytic agent for at least two days (i.e., 2 days or more) during a treatment window of at least 2 days and no more than about 13 days (i.e., 2-13 days); or administering the senolytic agent for at least two days (i.e., 2 days or more) during a treatment course of at least 2 days and no more than about 15 days (i.e., 2-15 days); or the senolytic agent is administered for at least two days (i.e., 2 days or more) during a treatment course of at least 2 days and not longer than about 16 days, 17 days, 18 days, 19 days, or 20 days (i.e., 2-16, 2-17, 2-18, 2-19, 2-20 days, respectively). In other embodiments, the senolytic agent can be administered for at least 3 days within a treatment course of at least 3 days and no longer than any number of days between 3 and 21 days; or for at least 4 days and no more than 4 to 21 days; or for at least 5 days within a treatment course of at least 5 days and no more than any number of days between 5 and 21 days; or for at least 6 days within a treatment course of at least 6 days and no more than any number of days between 6 and 21 days; or for at least 7 days within a treatment course of at least 7 days and no more than any number of days between 7 and 21 days; or for at least 8 or 9 days within a treatment course of at least 8 or 9 days, respectively, and no longer than any number of days between 8 or 9 days and 21 days, respectively; or for at least 10 days within a treatment course of at least 10 days and no more than any number of days between 10 and 21 days; or for at least 14 days within a treatment course of at least 14 days and no more than any number of days between 14 and 21 days; or for at least 11 or 12 days and not longer than any number of days between 11 or 12 days and 21 days, respectively; or for at least 15 or 16 days within a treatment course of at least 15 or 16 days, respectively, and no longer than any number of days between 15 or 16 days and 21 days, respectively. By way of further example, when the course of treatment is not longer than 14 days, the senolytic agent can be administered for at least 2,3, 4,5,6, 7,8, 9, 10, 11, 12, 13, and 14 days and for no longer than 14 days within a treatment window of at least 2,3, 4,5,6, 7,8, 9, 10, 11, 12, 13, and 14 days, respectively. When the course of treatment is not longer than 10 days, the senolytic agent can be administered for at least 2,3, 4,5,6, 7,8, 9, or 10 days within a treatment window of at least 2,3, 4,5,6, 7,8, 9, or 10 days and not longer than 10 days, respectively. Similarly, when the course of treatment is not longer than 7 days, the senolytic agent can be administered for at least 2,3, 4,5,6, or 7 days within a treatment window of at least 2,3, 4,5,6, or 7 days, respectively, and not longer than 7 days. In another example, when the treatment course is not longer than 5 days, the senolytic agent can be administered for at least 2,3, 4, or 5 days within a treatment window of at least 2,3, 4, or 5 days and not longer than 5 days, respectively.

For a three or more day treatment course, the dose of senolytic agent can be administered for a number of days less than the total number of days within the particular treatment window. By way of non-limiting example, when a course of treatment has a course of treatment of no more than 7, 10, 14, or 21 days, the senolytic agent can be administered for any number of days between 2 days and 7, 10, 14, or 21 days, respectively, and at any interval suitable for the particular disease being treated, the senolytic agent being administered, the health of the patient, and other relevant factors discussed in more detail herein. One skilled in the art will readily appreciate that when the senolytic agent is administered for two or more days within a treatment window, the agent can be delivered for the minimum number of days of the window, the maximum number of days of the window, or any number of days between the minimum and maximum.

In certain particular embodiments, the treatment course is one day or the treatment course is not more than 2,3, 4,5,6, 7,8, 9, 10, 11, 12, 13, or 14 days in length, which are examples of courses of treatment in which the senolytic agent is administered for two or more days within not more than (i.e., not longer than) 2,3, 4,5,6, 7,8, 9, 10, 11, 12, 13, or 14 days of treatment course, respectively. In certain other embodiments, the course of treatment is about 2 weeks (about 14 days or 0.5 months), about 3 weeks (about 21 days), about 4 weeks (about 1 month), about 5 weeks, about 6 weeks (about 1.5 months), about 2 months (or about 60 days), or about 3 months (or about 90 days). In particular embodiments, the course of treatment is a single daily administration of the senolytic agent. In other embodiments, for any course of treatment, the daily dose of senolytic agent can be as a single administration or the dose can be divided into 2,3, 4, or 5 separate administrations to provide a total daily dose of the agent.

As described herein, in certain particular embodiments, the course of treatment may have one or more intervals of one or more days during which the senolytic agent is not administered within a treatment window of two or more days during which the senolytic agent is administered. By way of non-limiting example only, when the treatment window is two to seven days, the first dose can be administered on the first day of the treatment window, and the second dose can be administered on the third day of the treatment course and the third dose can be administered on the seventh day of the treatment window. One skilled in the art will appreciate that different dosing schedules may be used during a particular treatment window. In other particular embodiments, the senolytic agent is administered continuously daily during the treatment course. The daily dose may be administered as a single dose or the daily dose may be divided into 2,3 or 4 or 5 separate administrations to provide a total daily dose of the senilising scavenger.

In certain embodiments, the course of treatment comprises a length of time that the senolytic agent is administered daily. In a particular embodiment, the senolytic agent is administered daily for 2 days. In another specific embodiment, the senolytic agent is administered daily for 3 days. In yet another specific embodiment, the senolytic agent is administered daily for 4 days. In a particular embodiment, the senolytic agent is administered daily for 5 days. In yet another specific embodiment, the senolytic agent is administered daily for 6 days. In another specific embodiment, the senolytic agent is administered daily for 7 days. In yet another specific embodiment, the senolytic agent is administered daily for 8 days. In yet another specific embodiment, the senolytic agent is administered daily for 9 days. In yet another specific embodiment, the senolytic agent is administered daily for 10 days. In yet another specific embodiment, the senolytic agent is administered daily for 11 days. In yet another specific embodiment, the senolytic agent is administered daily for 12 days. In yet another specific embodiment, the senolytic agent is administered daily for 13 days. In yet another specific embodiment, the senolytic agent is administered daily for 14 days. The treatment window (i.e., course) for each of the above examples is no longer than 2,3, 4,5,6, 7,8, 9, 10, 11, 12, 13, 14 days, respectively.

In other particular embodiments, the senolytic agent is administered every two days (i.e., every other day) for 3,4,5,6, 7,8, 9, 10, 11, 12, 13, or 14 days. In other particular embodiments, the senolytic agent is administered every three days (i.e., one day with the agent and then two days without the agent) for 4,5,6, 7,8, 9, 10, 11, 12, 13, or 14 days. In other particular embodiments, the senolytic agent can be administered every 2-3 days during a treatment window of 3,4,5,6, 7,8, 9, 10, 11, 12, 13, or 14 days. In other embodiments, every four days during a treatment course of 5,6, 7,8, 9, 10, 11, 12, 13, or 14 days; or every five days during a treatment course of 6,7, 8, 9, 10, 11, 12, 13, or 14 days. One skilled in the art can readily appreciate the minimum number of days of a treatment window when the senolytic agent is administered every six days, every seven days, etc., within the limited number of days of treatment window described herein.

In certain particular embodiments, the senolytic agent can be administered daily for a duration of longer than 14 days, and can be administered for at least 15, 16, 17, 18, 19, 20, or at least 21 days. In other particular embodiments, the senolytic agent can be administered daily for each of 15, 16, 17, 18, 19, 20, or 21 days. In another specific embodiment, the senolytic agent can be administered every two days during a treatment window of 15, 16, 17, 18, 19, 20, or 21 days. In another particular embodiment, the senolytic agent can be administered every three days during a treatment window of 15, 16, 17, 18, 19, 20, or 21 days. In other particular embodiments, the senolytic agent can be administered every 2-3 days during a treatment window of 15, 16, 17, 18, 19, 20, or 21 days. In other embodiments, every four days during a 15, 16, 17, 18, 19, 20, or 21 day treatment course; or every five days during a treatment course of 15, 16, 17, 18, 19, 20, or 21 days. One skilled in the art can readily understand the minimum number of days of a treatment window when the senolytic agent is administered every six days, every seven days, etc., within the limited number of days of treatment window described herein.

In another particular embodiment, the senolytic agent can be administered daily for a duration of longer than 14 days, and can be administered for at least 15, 16, 17, 18, 19, 20, or at least 21 days. In other particular embodiments, the senolytic agent may be administered daily for each of 15, 16, 17, 18, 19, 20, or 21 days. In another specific embodiment, the senolytic agent can be administered every two days during a treatment window of 15, 16, 17, 18, 19, 20, or 21 days. In another particular embodiment, the senolytic agent can be administered every three days during a treatment window of 15, 16, 17, 18, 19, 20, or 21 days. In other particular embodiments, the senolytic agent can be administered every 2-3 days during a treatment window of 15, 16, 17, 18, 19, 20, or 21 days. In other embodiments, every four days during a 15, 16, 17, 18, 19, 20, or 21 day treatment course; or every five days during a treatment course of 15, 16, 17, 18, 19, 20, or 21 days. One skilled in the art can readily understand the minimum number of days of a treatment window when the senolytic agent is administered every six days, every seven days, etc., within the limited number of days of treatment window described herein.

In another particular embodiment, the senolytic agent can be administered daily for a duration of longer than 14 days or 21 days during a treatment course, and can be administered during a treatment course of about one month, about two months, or about three months. In other particular embodiments, the senolytic agent can be administered daily during each of one, two, or three month treatment sessions. In another specific embodiment, the senolytic agent can be administered every two days during a treatment course of about one month, about two months, or about three months. In another particular embodiment, the senolytic agent can be administered every three days during a treatment course of about one month, about two months, or about three months. In other particular embodiments, the senolytic agent can be administered every 2-3 days during a treatment course of about one month, about two months, or about three months. In other embodiments, every four days may be followed during a treatment course of about one month, about two months, or about three months; or every five days during a treatment course of about one month, about two months, or about three months. One skilled in the art can readily appreciate the minimum number of days of a course of treatment when the senolytic agent is administered every six days, every seven days, etc., within the limited number of days of treatment window described herein.

By way of non-limiting example, a longer treatment window to reduce daily dose may be a treatment option for the subject. In other particular embodiments and by way of example, the stage or severity of the aging-related disease or disorder or other clinical factors may indicate that a longer term course of therapy may provide a clinical benefit. In certain embodiments, the treatment is performed for about 1-2 weeks (e.g., about 5-14 days), about 1-3 weeks (e.g., about 5-21 days), about 1-4 weeks (e.g., about 5-28 days), about 5-36 days, or about 5-42 days, 7-14 days, 7-21 days, 7-28 days, 7-36 days, or 7-42 days; or 9-14 days, 9-21 days, 9-28 days, 9-36 days, or 9-42 days, the senolytic agent is administered daily or optionally every other day (every two days) or every three days or at longer intervals (i.e., every four days, every five days, every six days). In certain other embodiments, the course of treatment is about 1-3 months. In a particular embodiment, the senolytic agent is administered daily for at least five days, and in another particular embodiment, the senolytic agent is administered daily for 5-14 days. In other particular embodiments, the senolytic agent is administered for at least seven days, e.g., 7-14, 7-21, 7-28, 7-36, or 7-42 days. In other particular embodiments, the senolytic agent is administered for at least nine days, e.g., 9-14 days, 9-21 days, 9-28 days, 9-36 days, or 9-42 days.

While a treatment regimen comprising administration of a senolytic agent provides a clinical benefit, as discussed herein and above, in other certain embodiments, the treatment regimen is repeated with a time interval between each treatment regimen during which no senolytic agent is administered (i.e., non-treatment interval, drug withdrawal treatment). A treatment cycle as described herein and in the art includes a treatment session followed by a non-treatment interval. The treatment cycle may be repeated as often as necessary. For example, the treatment cycle may be repeated at least once, at least twice, at least three times, at least four times, at least five times, or more frequently as desired. In certain particular embodiments, the treatment cycle is repeated once (i.e., administration of senolytic agent comprises 2 treatment cycles). In other certain embodiments, the treatment cycle is repeated two times or 3 or more times. Thus, in certain embodiments, one, two, three, four, five, six, seven, eight, nine, ten, or more treatment cycles of treatment with the senolytic agent are performed. In particular embodiments, the course of treatment or treatment cycle may be repeated, such as when the aging-related disease or disorder is relapsed, or when the symptoms or sequelae of the disease or disorder that are significantly alleviated by one course of treatment as described above have increased or are detectable, or when the symptoms or sequelae of the disease or disorder are exacerbated. In other embodiments, when administering a senolytic agent to a subject to prevent a senescence-associated disease or disorder (i.e., reduce the likelihood of occurrence or development of the senescence-associated disease or disorder) or delay the onset, progression, or severity of the senescence-associated disease or disorder, the subject can receive the senolytic agent over two or more treatment cycles. Thus, in certain embodiments, one treatment cycle is followed by a subsequent treatment cycle. Each treatment session of a treatment cycle or each treatment session of two or more treatment cycles is generally the same in terms of the duration and administration of the senolytic agent. In other embodiments, the duration and dosing of the senolytic agent during each treatment course of a treatment cycle can be adjusted as determined by one of skill in the medical arts, for example, according to the particular disease or disorder being treated, the senolytic agent being administered, the health of the patient, and other relevant factors discussed in more detail herein. Thus, the treatment course of the second or any subsequent treatment cycle may be shortened or lengthened as medically deemed necessary or prudent. In other words, as will be understood by those skilled in the art, each treatment session of the two or more treatment cycles is independent and the same or different; and each non-treatment interval of each treatment cycle is independent and the same or different.

As described herein, each course of treatment in a treatment cycle is separated by a time interval of days, weeks, or months (i.e., a non-treatment time interval or drug withdrawal interval; referred to herein as a non-treatment interval) that is not treated with senolytic agent. The non-treatment interval (e.g., days, weeks, months) between one treatment session and a subsequent treatment session is typically greater than the longest time interval (i.e., days) between any two days administered in a treatment session. For example, if a treatment course is no longer than 14 days and the agent is administered every other day during the treatment course, the non-treatment interval between two treatment courses is more than 2 days, such as 3,4,5,6, 7,8, 9, 10, 11, 12, 13, or 14 days or about 3 weeks, about 4 weeks, about 6 weeks, or about 2 months or longer as described herein. In particular embodiments, the non-therapeutic interval between two treatment sessions is about 5 days, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 6 weeks, about 2 months (8 weeks), about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months (about 1 year), about 18 months (about 1.5 years), or longer. In certain specific embodiments, the non-treatment interval is about 2 years or about 3 years. In certain specific embodiments, the non-treatment time interval is at least about 14 days, at least about 21 days, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, or at least about 1 year. In certain embodiments, a course of treatment is administered about every 14 days (i.e., about every 2 weeks) (i.e., 14 days without senolytic treatment), about every 21 days (i.e., about every 3 weeks), about every 28 days (i.e., about every 4 weeks), about every month, about every 36 days, about every 42 days, about every 54 days, about every 60 days or about every month (about every 30 days), about every two months (about every 60 days), about every quarter (about every 90 days), or about every half year (about every 180 days) (whether daily, every other day, every three days, or at other intervals between administrations within a course of treatment as described above (e.g., 1-14 days, 2-21 days, or 1-21 days)). In other certain embodiments, the course of treatment is administered every 28 days, every 36 days, every 42 days, every 54 days, every 60 days or every month (about every 30 days), every two months (about every 60 days), every quarter (about every 90 days), or every half year (about every 180 days), or about every year (about 12 months) (e.g., by way of non-limiting example, for at least one day or at least two days during a course of about 2-21 days, about 2-14 days, about 5-14 days, about 7-14 days, about 9-14 days, about 5-21 days, about 7-21 days, about 9-21 days). In other embodiments, the course of treatment is administered every 36 days, 42 days, 54 days, 60 days, or every month (about every 30 days), every two months (about every 60 days), quarterly (about every 90 days), or semiannually (about every 180 days) (by way of non-limiting example, for about 5-28 days, about 7-28 days, or about 9-28 days, whether daily, every other day, every three days, or at other intervals between administrations within the course of treatment). In other particular embodiments, a course of treatment is administered every 42 days, 54 days, 60 days, or every month (about every 30 days), every two months (about every 60 days), quarterly (about every 90 days), or semi-annually (about every 180 days), or about annually (about 12 months) (e.g., for about 5-36 days, 7-36 days, or 9-36 days, whether daily, every other day, every three days, or at other intervals between administrations within a course of treatment).

In particular embodiments, the course of treatment is one day, and the non-treatment intervals are at least about 14 days, about 21 days, about 1 month, about 2 months (8 weeks), about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months (about 1 year), about 18 months (about 1.5 years), or more. In certain other embodiments, the course of treatment is at least two days or at least 3 days and no longer than 10 days, and the non-treatment interval is at least about 14 days, about 21 days, about 1 month, about 2 months (8 weeks), about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months (about 1 year), about 18 months (about 1.5 years), or longer. In yet another embodiment, the treatment course is at least three days and no longer than 10 days, no longer than 14 days, or no longer than 21 days, and the non-treatment intervals are at least about 14 days, about 21 days, about 1 month, about 2 months (8 weeks), about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months (about 1 year), about 18 months (about 1.5 years), or longer. In yet another embodiment, a course of treatment is administered every 42 days, 60 days, or every month (about every 30 days), every two months (about every 60 days), quarterly (about every 90 days), or semiannually (about every 180 days), or about annually (about 12 months) (e.g., for about 5-42, 7-42, or 9-42 days, whether daily, every other day, every three days, or at other intervals between administrations within the course of treatment). In particular embodiments, the senolytic agent is administered daily for 5-14 days every 14 days (about every 2 weeks) or every 21-42 days. In another specific embodiment, the senolytic agent is administered daily for 5-14 days quarterly. In another specific embodiment, the senolytic agent is administered daily for 7-14 days every 21-42 days. In another specific embodiment, the senolytic agent is administered daily for 7-14 days quarterly. In other particular embodiments, the senolytic agent is administered daily every 21-42 days or every 9-14 days every quarter for 9-14 days. In other embodiments, the non-treatment interval may vary between treatment sessions. By way of non-limiting example, the non-treatment interval can be 14 days after the first treatment session and can be 21 days or more after the second, third, or fourth (or more) treatment session. In other particular embodiments, the senolytic agent is administered to a subject in need thereof once every 0.5-12 months. In certain other embodiments, the senilising scavenger is administered to a subject in need thereof every 4-12 months.

In certain embodiments, the senolytic agent is administered to the subject to reduce the likelihood or risk that the subject will develop a particular disorder, or to delay the onset of one or more symptoms of a senescence-associated disease or disorder. In certain embodiments, the senolytic agent is administered every 3,4,5,6, 7,8, 9, 10, 11, or 12 months for one or more days (e.g., any consecutive number of days including 2-3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, -17, -18, -19, -20, and 2-21 days). In particular embodiments, the senolytic agent is administered every 5 or 6 months for one or more days (e.g., any consecutive number of days including 1-9 days).

Without wishing to be bound by any particular theory, the periodic administration of senolytic agent kills newly formed senescent cells and thereby reduces (diminishes ) the total number of senescent cells accumulated in the subject. In another embodiment, the total number of senescent cells accumulated in the subject is reduced or inhibited by administering the senolytic agent once or twice a week or according to any of the other treatment courses described above. The total daily dose of senolytic agent upon daily administration may be delivered as a single dose or as multiple doses. In other certain particular embodiments, when the senolytic agent is administered for multiple cycles, the dose of senolytic agent administered per day may be less than the daily dose administered so long as the objective is to administer a single course of treatment.

In certain embodiments, a method for treating a senescence-associated disease or disorder comprises administering to a subject in need thereof a small molecule senolytic agent that selectively kills senescent cells; wherein the senescence-associated disease or disorder is not cancer, and wherein the senolytic agent is administered within one or two treatment cycles, typically two treatment cycles. In certain specific embodiments, the non-treatment interval is at least 2 weeks and each treatment course is no longer than 3 months.

Also provided herein are methods for selectively killing senescent cells, comprising contacting senescent cells with a senolytic agent described herein for a time and under conditions sufficient to kill senescent cells (i.e., to promote an interaction or in some way to cause the senescent cells to interact with the senolytic agent). In such embodiments, the agent selectively kills senescent cells over non-senescent cells (i.e., the agent selectively kills senescent cells as compared to killing non-senescent cells). In certain embodiments, the senescent cells to be killed are present in a subject (e.g., a human or non-human animal). The senolytic agent is administered to the subject according to the treatment cycle, treatment course, and non-treatment interval described above and herein.

In particular embodiments, a single (i.e., sole, separate) senolytic agent is administered to the subject to treat a senescence-associated disease or disorder. In certain embodiments, administration of a single senolytic agent may be sufficient and clinically beneficial for the treatment of a senescence-associated disease or disorder. Thus, in certain particular embodiments, the senolytic agent is administered as a monotherapy and is the sole (i.e., sole, separate) active agent administered to the subject to treat conditions and diseases. Administration of non-exclusionary drugs to a subject when the senolytic agent is administered as a monotherapy includes, by way of non-limiting example, drugs for other purposes such as palliative or comforting (e.g., aspirin, acetaminophen, ibuprofen, or a prescription analgesic; topical antipruritic drugs) or for treating various diseases or conditions, particularly if the other drug is not a senolytic agent, such as drugs for cholesterol reduction, statins, ocular moisturizers, and other such drugs familiar to those skilled in the medical arts.

In particular embodiments, if the senolytic agent is an MDM2 inhibitor, the MDM2 inhibitor is administered as a monotherapy (i.e., the only active therapeutic agent) and each course of treatment is at least 5 days long, during which the MDM2 inhibitor is administered for at least 5 days. In certain other embodiments, the MDM2 inhibitor is administered for at least 9 days. In a more specific embodiment, the MDM2 inhibitor is Nutlin-3 a.

The dosing regimen, course of treatment, and treatment cycle can be checked and modified or adjusted, continued or discontinued, as determined by one of skill in the art, based on the subject's response to the senolytic agent, the stage of the disease, the subject's overall health status, and other factors described herein and in the art.

As described herein, certain senolytic agents used in the methods may have been described as useful or potentially useful for treating cancer; however, in embodiments of the methods for treating a senescence-associated disorder or disease, the senolytic agent is administered in a manner that would be considered different from, and likely ineffective for, treating cancer. Thus, the methods described herein are useful for treating age-related conditions or diseases, but are not described as also useful as the first choice therapy for treating cancer (either alone or in combination with another chemotherapeutic agent or radiation therapy). In one embodiment, a method for treating a senescence-associated disease or disorder with a senolytic agent can comprise a reduced daily dose compared to the daily dose of agent required for cancer treatment. In another embodiment, a method for treating a senescence-associated disease or disorder with a senolytic agent described herein can comprise a cumulative dose that is reduced over a single treatment cycle compared to the cumulative dose of the agent required for cancer treatment. In yet another embodiment, a method for treating a senescence-associated disease or disorder with a senolytic agent described herein can comprise administering a cumulative dose of the agent that is reduced over a plurality of treatment cycles as compared to the dose of the agent required for the plurality of treatment cycles of cancer.

For example, in certain embodiments, when the senolytic agent is an agent that may be cytotoxic to cancer cells and that may be used in the oncology field in a manner to treat cancer (e.g., an MDM2 inhibitor (e.g., Nutlin-3 a; RG-7112), or an inhibitor of one or more BCL-2 anti-apoptotic protein family members and which inhibits at least Bcl-xL (e.g., ABT-263, ABT-737, WEHI-539, A-1155463)), methods for treating a senescence-associated disease or disorder comprise administering a senolytic agent in one or two or more treatment cycles, and the total dose of senolytic agent administered cumulatively during each treatment course, each treatment cycle, and/or over two or more treatment cycles is an amount less than the therapeutically effective amount of cancer. The amount of such senolytic agent administered to a subject over a given period of time (e.g., one week, two weeks, one month, six months, one year) to treat a senescence-associated disease or disorder can be a total amount that is reduced from about 20-fold to about 5000-fold compared to the total amount of the same agent administered to a subject receiving the agent for treating cancer. A fold reduction in the amount of (i.e., reduced amount of) a senolytic agent administered over a given time period (i.e., days, months, years) to treat a senescence-associated disease or disorder can be about 20 fold reduction, about 25 fold reduction, about 30 fold reduction, about 40 fold reduction, about 50 fold reduction, about 60 fold reduction, about 75 fold reduction, about 100 fold reduction, about 125 fold reduction, about 150 fold reduction, about 175 fold reduction, about 200 fold reduction, about 300 fold reduction, about 400 fold reduction, about 500 fold reduction, about 750 fold reduction, about 1000 fold reduction, about 1250 fold reduction, about 1500 fold reduction, about 1750 fold reduction, about 2000 fold reduction, about 2250 fold reduction, about 2500 fold reduction, about 2750 fold reduction, about 3000 fold reduction, about 3250 fold reduction, about 3500 fold reduction, about 3750 fold reduction, or a combination thereof, as compared to the amount of the agent administered to a subject over the same length of time to treat cancer, About 3000 fold reduction, about 3500 fold reduction, about 4000 fold reduction, about 4500 fold reduction, or about 5000 fold reduction. The lower dosage required to treat the aging-related disorder may also be attributed to the route of administration. For example, when the senolytic agent is used to treat a senolytic-related pulmonary disease or disorder (e.g., COPD, IPF), the senolytic agent can be delivered directly to the lung (e.g., by inhalation, by intubation, intranasally, or intratracheally), and a lower dose is required per day and/or per course of treatment than when the agent is administered orally. Moreover, as another example, when the senolytic agent is used to treat osteoarthritis or an aging-related skin disease or disorder, the senolytic agent can be delivered directly to the osteoarthritic joint (e.g., intra-articular, intradermal, topical, transdermal) or the skin (e.g., topical, subcutaneous, intradermal, transdermal), respectively, at a dose per day and/or per course of treatment that is lower than the dose at which the senolytic agent is administered orally. When the senolytic agent is delivered orally, for example, the dose of senolytic agent per day can be the same amount as administered to a patient to treat cancer; however, the amount of the agent delivered over a course or cycle of treatment is significantly less than the amount administered to a subject receiving an appropriate amount of the agent for treating cancer.

In certain embodiments, the methods described herein comprise the use of an amount of senolytic agent that is reduced compared to the amount that can be delivered systemically, e.g., orally or intravenously, to a subject receiving the senolytic agent when the agent is used to treat cancer. In certain particular embodiments, a method of treating a senescence-associated disease or disorder by selectively killing senescent cells comprises administering a senolytic agent at a dose that is at least 10% (i.e., one tenth), at least 20% (one fifth), 25% (one fourth), 30% -33% (about one third), 40% (two fifths), or at least 50% (half) of the dose administered to a subject having cancer during a course of treatment, a treatment cycle, or two or more treatment cycles that form a cancer treatment regimen (i.e., a regimen) to kill cancer cells. In other particular embodiments, the dose of the senolytic agent for use in the methods described herein is at least 60%, 70%, 80%, 85%, 90%, or 95% of the dose administered to a subject having cancer. Treatment regimens, including dosages of senolytic agents and administration schedules and modes useful for treating a senescence-associated disorder or disease, are also inadequate regimens for significant cytotoxicity against non-senescent cells.

In certain embodiments, a method for treating a non-cancer, senescence-associated disease or disorder comprises administering to a subject in need thereof a therapeutically effective amount of a small molecule senolytic agent that selectively kills senescent cells (i.e., selectively kills senescent cells relative to non-senescent cells or as compared to non-senescent cells), and that is cytotoxic to cancer cells, wherein the senolytic agent is administered for at least one treatment cycle comprising a treatment course followed by a non-treatment interval. The total dose of senolytic agent administered during the treatment course, and/or the total dose of senolytic agent administered during the treatment cycle, and/or the total dose of senolytic agent administered during two or more treatment cycles is an amount less than a cancer therapeutically effective amount. In certain embodiments, the senolytic agent is an inhibitor that inhibits at least a Bcl-2 anti-apoptotic protein family member of Bcl-xL; MDM2 inhibitors; or an Akt-specific inhibitor. Examples of such inhibitors are described herein. In other certain embodiments, the senolytic agent is administered as a monotherapy and is the sole active senolytic agent administered to the subject to treat the disease or disorder. Treatment courses and days between treatments are detailed herein.

In one embodiment, provided herein is a method for treating a senescence-associated disease or disorder, wherein the senescence-associated disease is not cancer and the method comprises administering to a subject in need thereof a senolytic agent or small molecule senolytic compound that selectively kills senescent cells, and the administration is for a short duration (e.g., shorter than the duration of a particular agent useful for treating cancer), such as one day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days. In these particular embodiments, the course of treatment for any number of days between 1-15 days is a single course of treatment and is not repeated. In another specific embodiment, the senolytic agent is administered for 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or 31 days (as a non-repeating single course of treatment).

In certain specific embodiments, the senolytic agent is ABT-263 (navitoclax). In some embodiments, navitoclax is administered in a treatment window comprising 21 days. In some embodiments, navitoclax is administered daily for 14 days, followed by 7 days of rest. In some embodiments, navitoclax is administered daily for 13 days, followed by 8 days of rest. In some embodiments, navitoclax is administered daily for 12 days, followed by 9 days of rest. In some embodiments, navitoclax is administered daily for 11 days, followed by 10 days of rest. In some embodiments, navitoclax is administered daily for 10 days, followed by 11 days of rest. In some embodiments, navitoclax is administered daily for 9 days, followed by 12 days of rest. In some embodiments, navitoclax is administered daily for 8 days, followed by 13 days of rest. In some embodiments, navitoclax is administered daily for 7 days, followed by 14 days of rest. In some embodiments, navitoclax is administered daily for 6 days, followed by 15 days of rest. In some embodiments, navitoclax is administered daily for 5 days, followed by 16 days of rest. In some embodiments, navitoclax is administered daily for 4 days, followed by 17 days of rest. In some embodiments, navitoclax is administered daily for 3 days, followed by 18 days of rest. In some embodiments, navitoclax is administered daily for 2 days, followed by 19 days of rest. In some embodiments, navitoclax is administered for 1 day, followed by 20 days of rest.

In some embodiments, navitoclax is administered daily at a dose of about 150mg to 325mg for 21 days. In some embodiments, navitoclax is administered daily at a dose of about 150mg to 300mg for 21 days. In some embodiments, navitoclax is administered daily at a dose of about 150mg to 275mg for 21 days. In some embodiments, navitoclax is administered daily at a dose of about 150mg to 250mg for 21 days. In some embodiments, navitoclax is administered daily at a dose of about 150mg to 225mg for 21 days. In some embodiments, navitoclax is administered daily at a dose of about 150mg to 200mg for 21 days. In some embodiments, navitoclax is administered daily at a dose of about 150mg to 175mg for 21 days. In some embodiments, navitoclax is administered daily at a dose of about 150mg for 21 days. In some embodiments, navitoclax is administered at a dose of about 125mg per day for 21 days. In some embodiments, navitoclax is administered daily at a dose of about 100mg for 21 days. In some embodiments, navitoclax is administered daily at a dose of about 75mg for 21 days. In some embodiments, navitoclax is administered daily at a dose of about 50mg for 21 days. In some embodiments, navitoclax is administered daily at a dose of about 25mg for 21 days.

In some embodiments, navitoclax is administered daily at a dose of about 150mg to 325mg for 14 days. In some embodiments, navitoclax is administered daily at a dose of about 150mg to 300mg for 14 days. In some embodiments, navitoclax is administered daily at a dose of about 150mg to 275mg for 14 days. In some embodiments, navitoclax is administered daily at a dose of about 150mg to 250mg for 14 days. In some embodiments, navitoclax is administered daily at a dose of about 150mg to 225mg for 14 days. In some embodiments, navitoclax is administered daily at a dose of about 150mg to 200mg for 14 days. In some embodiments, navitoclax is administered daily at a dose of about 150mg to 175mg for 14 days. In some embodiments, navitoclax is administered at a dose of about 150mg per day for 14 days. In some embodiments, navitoclax is administered at a dose of about 125mg per day for 14 days. In some embodiments, navitoclax is administered daily at a dose of about 100mg for 14 days. In some embodiments, navitoclax is administered at a dose of about 75mg per day for 14 days. In some embodiments, navitoclax is administered at a dose of about 50mg per day for 14 days. In some embodiments, navitoclax is administered at a dose of about 25mg per day for 14 days.

In some embodiments, navitoclax is administered daily at a dose of about 150mg to 325mg for 7 days. In some embodiments, navitoclax is administered daily at a dose of about 150mg to 300mg for 7 days. In some embodiments, navitoclax is administered at a dose of about 150mg to 275mg per day for 7 days. In some embodiments, navitoclax is administered daily at a dose of about 150mg to 250mg for 7 days. In some embodiments, navitoclax is administered daily at a dose of about 150mg to 225mg for 7 days. In some embodiments, navitoclax is administered daily at a dose of about 150mg to 200mg for 7 days. In some embodiments, navitoclax is administered daily at a dose of about 150mg to 175mg for 7 days. In some embodiments, navitoclax is administered daily at a dose of about 150mg for 7 days. In some embodiments, navitoclax is administered at a dose of about 125mg per day for 7 days. In some embodiments, navitoclax is administered daily at a dose of about 100mg for 7 days. In some embodiments, navitoclax is administered at a dose of about 75mg daily for 7 days. In some embodiments, navitoclax is administered daily at a dose of about 50mg for 7 days. In some embodiments, navitoclax is administered at a dose of about 25mg daily for 7 days. In other particular embodiments, the above dose is administered daily for 1, 2,3, 4, 5 or 6 days, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19 or 20 days.

In some embodiments, the senolytic agent is nutlin-3 a. In some embodiments, nutlin-3a is administered in a treatment window comprising 28 days. In some embodiments, nutlin-3a is administered daily for 10 days, followed by 18 days of rest. In some embodiments, nutlin-3a is administered daily for 9 days, followed by a 19 day off-course. In some embodiments, nutlin-3a is administered daily for 8 days, followed by 20 days of rest. In some embodiments, nutlin-3a is administered daily for 7 days, followed by 21 days of rest. In some embodiments, nutlin-3a is administered daily for 6 days, followed by a 22 day off-course. In some embodiments, nutlin-3a is administered daily for 5 days, followed by 23 days of rest. In some embodiments, nutlin-3a is administered daily for 4 days, followed by 24 days of rest. In some embodiments, nutlin-3a is administered daily for 3 days, followed by 25 days of rest. In some embodiments, nutlin-3a is administered daily for 2 days, followed by a 26 day off-course. In some embodiments, nutlin-3a is administered for 1 day, followed by 27 days of rest.

In some particular embodiments, at about 20mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, at about 19mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, at about 18mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, at about 17mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, at about 16mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, at about 15mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, at about 14mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, at about 13mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, at about 12mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, at about 11mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, the dosage is about 10mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, at about 9mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, at about 8mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, at about 7mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, at about 6mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, the amount is about 5mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, the amount of the active agent is about 4mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, the amount is about 3mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, at about 2mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, the dosage is about 1mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, the dosage is about 0.75mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, the amount is about 0.5mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, the dosage is about 0.25mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, the dosage is about 0.1mg/m per day²The dose of nutlin-3a is administered for 10 days. In some embodiments, the amount is about 0.01mg/m per day²The dose of nutlin-3a is administered for 10 days. In certain embodiments, nutlin-3a is administered at the above-described dose for 5, 6, 7, 8, 9, 11, 12, 13, or 14 days.

Age-related diseases or disorders

Provided herein are methods for treating conditions, diseases or disorders associated with, or caused by cellular aging, including age-related diseases or disorders in a subject in need thereof. A senescence-associated disease or disorder may also be referred to herein as a senescent cell-associated disease or disorder. Aging-related diseases or disorders include, for example, cardiovascular diseases and disorders, inflammatory diseases and disorders, autoimmune diseases and disorders, lung diseases and disorders, ocular diseases and disorders, metabolic diseases and disorders, neurological diseases and disorders (e.g., neurodegenerative diseases and disorders); age-related diseases and disorders caused by aging; a skin condition; age-related diseases; skin diseases and disorders; and transplantation-related diseases and disorders. Aging is characterized by a gradual loss or deterioration of function at the molecular, cellular, tissue and biological level. Age-related degeneration causes well-recognized pathologies such as muscle atrophy, atherosclerosis and heart failure, osteoporosis, pulmonary valve insufficiency, renal failure, neurodegeneration (including macular degeneration, alzheimer's disease, and parkinson's disease), and many others. Although different mammalian species vary in susceptibility to particular age-related pathologies, in general, age-related pathologies typically rise approximately exponentially starting from the approximate midpoint of the species-specific lifespan (e.g., 50-60 years for humans) (see, e.g., Campisi, Annu. Rev. physiol.75:685-705 (2013); Naylor et al, Clin. Pharmacol.Ther.93:105-16 (2013)).

Examples of senescence-associated conditions, disorders, or diseases that can be treated according to the methods described herein by administering any of the senolytic agents described herein include cognitive diseases (e.g., Mild Cognitive Impairment (MCI), Alzheimer's disease, and other dementias; Huntington's disease); cardiovascular disease (e.g., atherosclerosis, diastolic dysfunction, aortic aneurysm, angina, arrhythmia, cardiomyopathy, congestive heart failure, coronary artery disease, myocardial infarction, endocarditis, hypertension, carotid artery disease, peripheral vascular disease, cardiac stress resistance, myocardial fibrosis); metabolic diseases and disorders (e.g., obesity, diabetes, metabolic syndrome); motor function diseases and disorders (e.g., Parkinson's disease, Motor Neuron Dysfunction (MND); Huntington's disease); cerebrovascular disease; emphysema; osteoarthritis; benign prostatic hyperplasia; lung diseases (e.g., idiopathic pulmonary fibrosis, Chronic Obstructive Pulmonary Disease (COPD), emphysema, obstructive bronchiolitis, asthma); inflammatory/autoimmune diseases and disorders (e.g., osteoarthritis, eczema, psoriasis, osteoporosis, mucositis, graft-related diseases and disorders); eye diseases or disorders (e.g., age-related macular degeneration, cataracts, glaucoma, vision loss, presbyopia); diabetic ulcers; transferring; chemotherapy side effects, radiotherapy side effects; age-related diseases and disorders (e.g., kyphosis, renal insufficiency, weakness, hair loss, hearing impairment, muscle fatigue, skin conditions, muscle atrophy, and disc herniation) and other age-related diseases caused by aging (e.g., diseases/disorders resulting from irradiation, chemotherapy, smoking tobacco, eating a high fat/high sugar diet, and environmental factors); healing the wound; a skin nevus; fibrotic diseases and disorders (e.g., cystic fibrosis, renal fibrosis, liver fibrosis, pulmonary fibrosis, oral submucosal fibrosis, myocardial fibrosis, and pancreatic fibrosis). In certain embodiments, any one or more of the diseases or disorders described above or herein may be excluded.

In more particular embodiments, methods are provided for treating a disease or disorder associated with aging by administering a senolytic agent to kill aging cells (i.e., established aging cells) associated with the disease or disorder in a subject having the disease or disorder, wherein the disease or disorder is osteoarthritis; idiopathic pulmonary fibrosis; chronic Obstructive Pulmonary Disease (COPD) or atherosclerosis.

Subjects (i.e., patients, individuals (human or non-human animals)) that may benefit from use of the methods described herein that include administration of senolytic agents include those subjects that may also have cancer. A subject treated by these methods may be considered to be in partial or complete remission (also referred to as cancer remission). As discussed in detail herein, the senolytic agent used in the method of selectively killing senescent cells is not intended for use as a treatment for cancer, that is, is not used in a manner that kills or destroys cancer cells in a statistically significant manner. Thus, the methods disclosed herein do not encompass the use of senolytic agents in a manner that would be considered the first choice therapy for treating cancer. Although senolytic agents are not used alone or with other chemotherapeutic or radiotherapeutic agents in a manner sufficient to be considered a first choice cancer therapy, the methods and senolytic agents described herein can be used in a manner useful for inhibiting metastasis (e.g., a short course of treatment). In other certain embodiments, the subject to be treated with the senolytic agent does not have cancer (i.e., the subject is not diagnosed as having cancer by a person of skill in the medical arts).

Cardiovascular diseases and disorders.In another embodiment, the aging-related disease or condition treated by the methods described herein is a cardiovascular disease. The cardiovascular disease can be angina pectoris, arrhythmia, atherosclerosis, cardiomyopathy, congestive heart failure, Coronary Artery Disease (CAD), carotid artery disease, endocarditis, heart attack (coronary thrombosis, myocardial infarction [ MI)]) Any one or more of high blood pressure, hypertension, aortic aneurysm, cerebral aneurysm, myocardial fibrosis, diastolic dysfunction, hypercholesterolemia/hyperlipidemia, mitral valve prolapse, peripheral vascular disease (e.g., Peripheral Arterial Disease (PAD)), cardiac stress resistance, and stroke.

In certain embodiments, methods are provided for treating aging-related cardiovascular diseases associated with or caused by arteriosclerosis (i.e., arteriosclerosis). The cardiovascular disease can be atherosclerosis (e.g., Coronary Artery Disease (CAD) and carotid artery disease); angina, congestive heart failure, and peripheral vascular disease (e.g., Peripheral Arterial Disease (PAD)). Methods for treating cardiovascular disease associated with or caused by arteriosclerosis may reduce the likelihood of developing high blood pressure (high blood pressure)/hypertension (hypertension), angina, stroke, and heart attack (i.e., coronary thrombosis, Myocardial Infarction (MI)). In certain embodiments, methods are provided for stabilizing atherosclerotic plaques in a blood vessel (e.g., artery) of a subject, thereby delaying the onset or reducing the likelihood of a thrombotic event such as stroke or MI. In certain embodiments, these methods comprising administering an senolytic agent reduce (i.e., cause to reduce) the lipid content of an atherosclerotic plaque in a blood vessel (e.g., artery) of the subject, and/or increase the thickness of the fibrous cap (i.e., result in enhancing, strengthening, or promoting thickening of the fibrous cap).

Atherosclerosis is characterized by a patchy intimal plaque (atheroma) that encroaches on the lumen of medium and large arteries; the plaque contains lipids, inflammatory cells, smooth muscle cells, and connective tissue. Atherosclerosis can affect the aorta and the middle arteries (including coronary, carotid and cerebral), the aorta and its branches, and the major arteries at the extremities of the bones. Atherosclerosis is characterized by a patchy intimal plaque (atheroma) that encroaches on the lumen of medium and large arteries; the plaque contains lipids, inflammatory cells, smooth muscle cells, and connective tissue.

In one embodiment, a method is provided for inhibiting the formation of (or reducing, diminishing, causing the reduction of the formation of) atherosclerotic plaques by administering a senolytic agent. In other embodiments, methods for reducing (reducing, decreasing) the amount (i.e., level) of plaque are provided. For example, the amount of plaque reduction in a vessel (e.g., artery) can be determined by a reduction in the surface area of the plaque or by a reduction in the extent and extent (e.g., percentage) of vessel (e.g., artery) occlusion, which can be determined by angiography or other visualization methods used in the cardiovascular field. Also provided herein are methods for enhancing (or improving, promoting, enhancing stability) the stability of an atherosclerotic plaque present in one or more blood vessels (e.g., one or more arteries) of a subject comprising administering to the subject any one of the senolytic agents described herein.

Atherosclerosis is commonly referred to as "hardening" or fouling of the arteries, and it is caused by the formation of multiple atheromatous plaques within the arteries. Atherosclerosis (also referred to herein and in the art as arteriosclerotic vascular disease or ASVD) is a form of arteriosclerosis with thickened arterial walls. Symptoms develop when the growth or rupture of the plaque reduces or impedes blood flow; and the symptoms may vary depending on which artery is affected. Atherosclerotic plaques may be stable or unstable. Stable plaques degenerate, remain static, or slowly, sometimes over decades of growth until they can cause stenosis or obstruction. Unstable plaques are susceptible to spontaneous erosion, fissures or rupture, which causes acute thrombosis, obstruction and infarction, as early as they cause a stenosis that is hemodynamically significant. Most clinical events are caused by unstable plaques, which do not appear to be severe on angiography; plaque stabilization may therefore be a method of reducing morbidity and mortality. Plaque rupture or erosion can lead to major Cardiovascular events such as acute coronary syndromes and stroke (see, e.g., Du et al, BMC Cardiovascular Disorders 14:83 (2014); Grimm et al, Journal of Cardiovascular Magnetic Resonance 14:80 (2012)). Disrupted plaques were found to have a higher content of lipids, macrophage cells, and a thinner fibrous cap than intact plaques (see, e.g., Felton et al, Arteriosclerosis, Thrombosis, and Vascular Biology 17:1337-45 (1997)).

Atherosclerosis is a syndrome of invading arterial vessels mainly caused by a chronic inflammatory response of leukocytes in the arterial wall. The syndrome is promoted by low density lipoproteins (LDL, plasma proteins carrying cholesterol and triglycerides) in the absence of adequate removal of fat and cholesterol from macrophages by functional High Density Lipoproteins (HDL). The earliest visible lesion of atherosclerosis is the "fatty streak", which is the accumulation of lipid-laden foam cells in the intimal layer of the artery. Atherosclerosis is characterized by atherosclerotic plaques, which are the evolution of the fatty streak and have three major components: lipids (e.g., cholesterol and triglycerides); inflammatory cells and smooth muscle cells; and connective tissue matrix that may contain thrombi and calcium deposits at various stages of organization. Calcium and other crystalline components (e.g., microcalcifications) from dead cells can be found in the outermost and oldest plaques. Microcalcification and properties associated therewith are also thought to contribute to plaque instability by increasing plaque stress (see, e.g., Bluestein et al, J.Biomech.41(5):1111-18 (2008); Cilla et al, Journal of Engineering in Medicine227:588-99 (2013)). The fatty streak reduces the elasticity of the arterial wall, but may not affect blood flow for years because the arterial muscle wall adapts by enlarging at the site of the plaque. Lipid-rich atheromas carry an increased risk of plaque rupture and thrombosis (see, e.g., Felton et al, supra; Fuster et al, J.Am.Coll.Cardiol.46:1209-18 (2005)). It was reported that the lipid core showed the highest thrombogenic activity among all plaque components (see, e.g., Fernandez-Ortiz et al, J.Am.Coll.Cardiol.23:1562-69 (1994)). In the major arteries in advanced disease, the wall sclerosis (stilbening) can also eventually increase the pulse pressure.

Vulnerable plaques can lead to thrombotic events (Stroke or MI) and are sometimes described as a large soft lipid pool covered by a thin fibrous cap (see, e.g., Li et al, Stroke 37:1195-99 (2006); Trivedi et al, neuro biology 46:738-43 (2004)). Advanced atherosclerotic plaques are characterized by irregular thickening of the intima of the artery by inflammatory cells, extracellular lipids (atheroma) and fibrous tissue (cirrhosis) (see, e.g., Newby et al, cardiovasc. res.345-60 (1999)). Fibrous cap formation is thought to occur due to migration and proliferation of vascular smooth muscle cells and due to matrix deposition (see, e.g., Ross, Nature 362:801-809 (1993); Sullivan et al, J.angiology at dx.doi.org/10.1155/2013/592815 (2013)). A thin fibrous cap contributes to the instability of the plaque and increases the risk of rupture (see, e.g., Li et al, supra).

Both pro-inflammatory macrophages (M1) and anti-inflammatory macrophages (M2) were found in the arteriosclerotic plaques. The contribution of these two types to plaque instability is the subject of active research, with results indicating that increased levels of the M1 type relative to the M2 type correlate with increased instability of plaque (see, e.g., Medbury et al, int.angiol.32:74-84 (2013); Lee et al, am.j.clin.pathol.139:317-22 (2013); Martinet et al, cir.res.751-53 (2007)).

Subjects with cardiovascular disease can be identified using standard diagnostic methods known in the art of cardiovascular disease. Generally, diagnosis of atherosclerosis and other cardiovascular diseases is based on the patient's symptoms (e.g., chest pain or tightness (angina), numbness or weakness in arms and legs, difficulty speaking or speech loss, facial muscle drop, leg pain, hypertension, renal failure and/or erectile dysfunction), medical history and/or physical examination. The diagnosis may be confirmed by angiography, ultrasonography, or other imaging examination. Subjects at risk of developing cardiovascular disease include those with any one or more of the susceptibility factors, such as a family history of cardiovascular disease, as well as those with other risk factors (i.e., susceptibility factors) such as hypertension, dyslipidemia, high cholesterol, diabetes, obesity and smoking, sedentary lifestyle, and hypertension. In a certain embodiment, the cardiovascular disease that is a senescent cell-related disease/disorder is atherosclerosis.

One of skill in the medical and clinical arts can readily determine the efficacy of one or more senolytic agents for the treatment or prevention (i.e., reducing or diminishing the likelihood of occurrence or development) of a cardiovascular disease (e.g., atherosclerosis). One or any combination of diagnostic methods (including physical examination, assessment and monitoring of clinical symptoms) and performing analytical tests and methods described herein and practiced in the art (e.g., angiography, electrocardiography, stress tests, non-stress tests) can be used to monitor the health condition of a subject. The therapeutic effect of a senolytic agent or a pharmaceutical composition comprising a senolytic agent can be analyzed using techniques known in the art, such as comparing the symptoms of a subject having or at risk of cardiovascular disease who has received the treatment to those of an untreated or placebo-treated patient.

Inflammatory and autoimmune diseases and disorders. In certain embodiments, the senescence-associated disease or disorder is an inflammatory disease or disorder that can be treated or prevented (i.e., reduce the likelihood of occurrence) according to the methods described herein comprising administration of a senolytic agent, such as, by way of non-limiting example, osteoarthritis. Other inflammatory or autoimmune diseases or conditions that may be treated by administration of senolytic agents such as the inhibitors and antagonists described herein include osteoporosis, psoriasis, oral mucositis, rheumatoid arthritis, inflammatory bowel disease, eczema, kyphosis, herniated intervertebral discs, as well as pulmonary diseases, COPD, and idiopathic pulmonary fibrosis.

Osteoarthritis degenerative joint disease is characterized by fibrillation of cartilage at sites of high mechanical stress, bone sclerosis, and thickening of the synovium and joint capsule. Fibrillation is the local surface tissue destruction (disorganization) of the breakdown of the superficial layer of cartilage (splitting). The early breakdown is tangential to the cartilage surface, along the axis of the main collagen bundle. Collagen within the cartilage becomes disorganized and proteoglycans are lost to the cartilage surface. Without the protective and lubricating effect of proteoglycans in the joints, collagen fibers become susceptible to degradation and subsequent mechanical destruction. The risk factors for developing osteoarthritis include increased age, obesity, previous joint injury, joint overuse, thigh muscle weakness, and inheritance. It is a common cause of chronic disability in the elderly. Symptoms of osteoarthritis include pain or joint stiffness (particularly the hip, knee and lower back) after inactivity or overuse; stiffness after rest that disappears after exercise; and more severe pain after activity or near the end of the day. Osteoarthritis also often affects the neck, the small finger joints, the base of the thumb, ankle and big toe.

Chronic inflammation is considered to be a major age-related factor leading to osteoarthritis. Joint overuse and obesity, in combination with aging, appear to contribute to osteoarthritis.

Surprisingly, by selectively killing senescent cells, senolytic agents prevent (i.e., reduce the likelihood of occurrence), reduce or inhibit proteoglycan loss or erosion in joints, reduce inflammation in affected joints, and promote (i.e., stimulate, enhance, induce) the production of collagen (e.g., type 2 collagen). Removal of senescent cells results in a reduction in the amount (i.e., level) of inflammatory factors such as IL-6 produced in the joints, and thus inflammation is reduced. Provided herein are methods of treating osteoarthritis, selectively killing senescent cells in an osteoarthritic joint of a subject, and/or inducing collagen (such as type 2 collagen) production in a joint of a subject in need thereof, by administering to the subject at least one senolytic agent (which may be combined with at least one pharmaceutically acceptable excipient to form a pharmaceutical composition). Senolytic agents can also be used to reduce (inhibit, reduce) the production of metalloprotease 13 (MMP-13) that degrades collagen in the joint, and to restore the proteoglycan layer or inhibit the loss and/or degradation of the proteoglycan layer. Thus, treatment with a senolytic agent also prevents (i.e., reduces the likelihood of occurrence of), inhibits or reduces erosion, or slows (i.e., reduces the rate) erosion of bone. As described in detail herein, in certain embodiments, the senolytic agent is administered directly to the osteoarthritic joint (e.g., via intraarticular, topical, transdermal, intradermal, or subcutaneous delivery). Treatment with senolytic agents may also restore, improve, or inhibit deterioration of joint strength. In addition, methods involving administration of senolytic agents can alleviate joint pain, and thus are useful for pain control in osteoarthritic joints.

One skilled in the medical and clinical arts can readily determine the effectiveness of one or more senolytic agents for treating or preventing osteoarthritis in a subject and the monitoring of a subject receiving one or more senolytic agents. One or any combination of diagnostic methods may be used to monitor the health status of a subject, including physical examination (e.g., determining tenderness, swelling, or redness of the affected joint), evaluation and monitoring of clinical symptoms (e.g., pain, stiffness, mobility), performance of the analytical tests and methods described herein and practiced in the art (e.g., determining levels of inflammatory or chemotactic factors; X-ray images used to determine cartilage loss as shown by narrowing the gap between bones in a joint; Magnetic Resonance Imaging (MRI) providing detailed images of bones and soft tissues including cartilage). The therapeutic effect of one or more senolytic agents can be analyzed by comparing the symptoms of a patient who has received treatment and is suffering from or at risk of an inflammatory disease or disorder, such as osteoarthritis, to the symptoms of a patient who has not received such treatment or who has received placebo treatment.

In certain embodiments, the senolytic agent can be used to treat and/or prevent (i.e., reduce or reduce the likelihood of occurrence of) Rheumatoid Arthritis (RA). Rheumatoid Arthritis (RA), characterized by dysregulation of innate and adaptive immune responses, is an autoimmune disease and its incidence increases with age. Rheumatoid arthritis is a chronic inflammatory condition that commonly affects the facet joints in the hands and feet. Osteoarthritis is caused at least in part by wear of the joint, while rheumatoid arthritis affects the lining of the joint, resulting in painful swelling that can cause bone erosion and joint deformity. RA can also sometimes affect other organs of the body, such as the skin, eyes, lungs, and blood vessels. RA can occur in subjects of any age; however, RA usually begins to develop after age 40. This condition is more common in women. In certain embodiments of the methods described herein, RA is excluded.

Chronic inflammation can also lead to other age-related or aging-related diseases and disorders, such as kyphosis and osteoporosis. Kyphosis is a severe curvature of the spine and is often observed with normal and premature aging (see, e.g., Katzman et al, (2010) j.ortho. Sports. ther.40: 352-. Age-related kyphosis usually occurs after the osteoporotic weakening of the spinal bone reaches the point where it ruptures and compresses. A small variety of kyphosis is found in infants or adolescents. Severe kyphosis can affect the lungs, nerves and other tissues and organs, causing pain and other problems. Kyphosis is associated with cellular senescence. Ability of senolytic agents to treat kyphosisCharacterization can be determined in preclinical animal models used in the art. By way of example, TTD mice develop kyphosis (see, e.g., de Boer et al, (2002) Science296: 1276-; other mice that can be used include BubR1^H/HMice, which are also known to develop kyphosis (see, e.g., Baker et al, (2011) Nature 479: 232-36). Formation of kyphosis was visualized over time. The level of senescent cells reduced by treatment with a senolytic agent can be determined by detecting the presence of one or more senescent cell-associated markers via, for example, SA- β -Gal staining.

Osteoporosis is a progressive bone disease characterized by a decrease in bone mass and density, which can lead to an increased risk of fracture. Bone Mineral Density (BMD) decreases, bone microarchitecture deteriorates, and the amount and diversity of proteins in bone changes. Osteoporosis is usually diagnosed and monitored by bone mineral density detection. Postmenopausal women or estrogen-reduced women are at the greatest risk. Both men and women over the age of 75 are at risk, but women are twice as likely to develop osteoporosis as men. The level of senescent cells reduced by treatment with a senolytic agent can be determined by detecting the presence of one or more senescent cell-associated markers via, for example, SA- β -Gal staining.

In other embodiments, inflammatory/autoimmune disorders that can be treated or prevented (i.e., have a reduced likelihood of occurrence) by the senolytic agents described herein include Irritable Bowel Syndrome (IBS) and inflammatory bowel disease, such as ulcerative colitis and crohn's disease. Inflammatory Bowel Disease (IBD) involves chronic inflammation of some or all of the digestive tract. In addition to the life-threatening complications caused by IBD, the disease can be painful and debilitating. Ulcerative colitis is an inflammatory bowel disease that causes chronic inflammation in a portion of the digestive tract. Symptoms typically develop over time rather than suddenly. Ulcerative colitis generally affects only the innermost lining of the large intestine (colon) and rectum. Crohn's disease is an inflammatory bowel disease that causes inflammation anywhere along the lining of the digestive tract and generally extends deep into the affected tissues. This can lead to abdominal pain, severe diarrhea and malnutrition. Inflammation caused by crohn's disease can affect different areas of the digestive tract. Diagnosis and monitoring of the disease is performed according to methods and diagnostic tests routinely practiced in the art, including blood tests, colonoscopy, sigmoidoscopy, barium enema, CT scan, MRI, endoscopy, and small intestine imaging.

In other embodiments, the methods described herein may be useful for treating a subject having a disc herniation. Subjects with herniated discs exhibit increased presence of cellular senescence in the blood and vessel walls (see, e.g., Roberts et al, (2006) eur. spine j.15 Suppl 3: S312-316). Symptoms of disc herniation may include pain, numbness or tingling, or weakness of the arms or legs. Elevated levels of proinflammatory molecules and matrix metalloproteinases have also been found in aging and degenerating disc tissue, suggesting their effect on aging cells (see, e.g., Chang-Qing et al, (2007) aging Res. Rev.6: 247-61). An animal model can be used to characterize the effectiveness of the senolytic agent in treating a disc herniation; degeneration of the disc is induced in mice by compression and increased disc strength (see, e.g., Lotz et al, (1998) Spine (Philadelphia Pa.1976).23: 2493-.

Other inflammatory or autoimmune diseases that can be treated or prevented (i.e., reduce the likelihood of occurrence) by the use of senolytic agents include eczema, psoriasis, osteoporosis, and pulmonary diseases (e.g., Chronic Obstructive Pulmonary Disease (COPD), Idiopathic Pulmonary Fibrosis (IPF), asthma), inflammatory bowel disease, and mucositis, including oral mucositis, which in some cases is induced by radiation. Certain fibrotic or fibrotic conditions of organs such as kidney fibrosis, liver fibrosis, pancreatic fibrosis, cardiac fibrosis, skin wound healing and oral submucosa fibrosis can be treated with senolytic agents.

In certain embodiments, the senescent cell-related disorder is a dermatitis disorder such as, by way of non-limiting example, psoriasis and eczema, which can be treated or prevented (i.e., reduced in likelihood of occurrence) according to the methods described herein comprising administration of a senolytic agent. Psoriasis is characterized by an abnormally large and rapid growth of the epidermal layer of the skin. The diagnosis of psoriasis is usually based on the appearance of the skin. The typical skin characteristic of psoriasis is red scaly patches, papules, or patches of skin that may be painful and itchy. In psoriasis, cutaneous and systemic overexpression of IL-6, a key component of a number of pro-inflammatory cytokines such as SASP, is observed. Eczema is an inflammation of the skin characterized by redness, swelling of the skin, itching and dryness, scabbing, flaking, blistering, cracking, oozing or bleeding. One skilled in the medical or clinical arts can readily determine the effectiveness of senolytic agents for the treatment of psoriasis and eczema and the monitoring of a subject receiving such senolytic agents. One or any combination of diagnostic methods include physical examination (e.g., skin appearance), assessment or monitoring of clinical symptoms (e.g., itch, swelling, and pain), and performance of the assays and methods described herein and practiced in the art (i.e., determining the level of proinflammatory cytokines).

Other immune diseases or conditions that can be treated or prevented (i.e., have a reduced likelihood of occurrence) using senolytic agents include conditions resulting from the host's immune response to an organ transplant (e.g., a kidney, bone marrow, liver, lung, or heart transplant), such as rejection of the transplanted organ. Senolytic agents can be used to treat or reduce the likelihood of graft versus host disease.

Pulmonary diseases and disorders.In one embodiment, a method of treating or preventing (i.e., reducing the likelihood of occurrence of) a senescence-associated disease or disorder, which is a pulmonary disease or disorder, by killing senescent cells (i.e., defined senescent cells) associated with the disease or disorder in a subject having the disease or disorder by administering a senolytic agent is provided. Aging-related lung diseases and disorders include, for example, Idiopathic Pulmonary Fibrosis (IPF), Chronic Obstructive Pulmonary Disease (COPD), asthma, cystic fibrosis, bronchiectasis, and emphysema.

COPD is a lung disease defined as persistent poor airflow, which is caused by destruction of lung tissue (lung emphysema) and dysfunction of the small airways (obstructive bronchiolitis). The major symptoms of COPD include shortness of breath, asthma, chest tightness, chronic cough and excessive sputum production. Elastase from cigarette smoke-activated neutrophils and macrophages breaks down the extracellular matrix of the alveolar structure, resulting in increased air space and loss of respiratory capacity (see, e.g., Shapiro et al, am.j.respir.cell mol.biol.32, 367-372 (2005)). COPD is most commonly caused by tobacco smoke (including cigarette smoke, cigar smoke, second hand smoke, pipe smoke), occupational exposure (e.g., exposure to dust, smoke or smoke) and pollution, occurring over decades, thereby suggesting that aging is a risk factor for developing COPD.

The processes involved in causing lung injury include, for example, oxidative stress generated by high concentrations of free radicals in tobacco smoke; cytokine release due to inflammatory response to irritants in the airways; and damage by tobacco smoke and free radicals to protease-resistant enzymes, causing damage by proteases to the lungs. Genetic predisposition may also contribute to the disease. In about 1% of people with COPD, the disease is caused by genetic disorders that lead to low levels of alpha-1-antitrypsin production in the liver. The enzyme is usually secreted into the bloodstream to help protect the lungs.

Pulmonary fibrosis is a chronic and progressive lung disease characterized by cirrhosis and scarring of the lungs, which can lead to respiratory failure, lung cancer, and heart failure. Fibrosis is associated with epithelial repair. Fibroblasts are activated, extracellular matrix protein production is increased, and transdifferentiation (transdifferentiation) to contractile myofibroblasts contributes to wound contraction. The temporary matrix occludes the injured epithelium and provides a scaffold for epithelial cell migration, involving epithelial-mesenchymal transition (EMT). Blood loss associated with epithelial injury induces platelet activation, growth factor production, and an acute inflammatory response. Typically, the epithelial barrier heals and the inflammatory response subsides. However, in fibrotic diseases, the fibroblast response continues, resulting in unreduced wound healing. The formation of a fibroblast foci is a characteristic of the disease, reflecting the location of persistent fibrogenesis. As its name implies, the etiology of IPF is unknown. Cellular senescence has been shown to be associated with IPF based on the increasing incidence of disease with age and the observation that lung tissue in IPF patients is enriched with SA- β -Gal-positive cells and contains elevated levels of the senescence marker p21 (see, e.g., Minagawa et al, am.j.physiol.lungcell.mol.physiol.300: L391-L401 (2011; see also, e.g., Naylor et al, supra). Short telomeres are a common risk factor for both IPF and cell senescence (see, e.g., Alder et al, proc.natl.acad.sci.usa 105: 13051-56 (2008)). Without wishing to be bound by theory, the contribution of cellular senescence to IPF is suggested according to reports that the SASP components of senescent cells, such as IL-6, IL-8 and IL-1 β, promote differentiation of fibroblasts into myofibroblasts and epithelial-mesenchymal transition, leading to extensive remodeling of the extracellular matrix of the alveolar and interstitial spaces (see, e.g., Minagawa et al, supra).

Subjects at risk of developing pulmonary fibrosis include those exposed to environmental or occupational contaminants, such as asbestos lung and silicosis; a subject smoking; subjects with some typical connective tissue diseases such as rheumatoid arthritis, SLE and scleroderma; subjects with other diseases that involve connective tissue, such as sarcoidosis and wegener's granuloma; a subject having an infection; subjects using certain drugs (e.g., amiodarone, bleomycin, busulfan, methotrexate and nitrofurantoin); a subject receiving radiation therapy for the chest; and those subjects whose family members have pulmonary fibrosis.

Symptoms of COPD may include any of the following: shortness of breath, particularly during physical activity; asthma; chest distress; the first thing in the morning must clear the throat due to excess mucus in the lungs; chronic cough producing sputum that may be clear, white, yellow or greenish; lips or nails blue (cyanosis); frequent respiratory infections; lassitude; unintended weight loss (observed later in the disease). A subject with COPD may also experience an exacerbation during which symptoms worsen and persist for days or longer. Symptoms of pulmonary fibrosis are known in the art and include shortness of breath, particularly when exercising; dry cough; fast and shallow breathing; gradual unintended weight loss; fatigue; joint and muscle pain; and clubbing (widening and rounding of the tip of the finger or toe).

Subjects suffering from COPD or pulmonary fibrosis can be identified using standard diagnostic methods routinely practiced in the art. The effect of one or more senolytic agents administered to a subject suffering from or at risk of developing a pulmonary disease can be monitored using methods commonly used for diagnostics. Generally, one or more of the following checks or tests may be performed: physical examination, patient history, patient family history, chest x-ray, pulmonary function tests (e.g., spirometry), blood tests (e.g., arterial blood gas analysis), bronchoalveolar lavage, lung biopsy, CT scan, and exercise tests.

Other pulmonary diseases or conditions that can be treated by the use of senolytic agents include, for example, emphysema, asthma, bronchiectasis, and cystic fibrosis (see, e.g., Fischer et al, Am J Physiol Lung Cell mol physiol.304(6): L394-400 (2013)). These diseases can also be exacerbated by tobacco smoke (including cigarette smoke, cigar smoke, second hand smoke, pipe smoke), occupational exposure (e.g., exposure to dust, smoke, or smoke), infection, and/or pollutants that induce cellular entry into aging and thereby cause inflammation. Emphysema is sometimes considered a subgroup of COPD.

Bronchiectasis is caused by airway damage that causes the airway to widen and become flaccid and scarred. Bronchiectasis is often caused by medical conditions that damage the airway wall or inhibit the airway from clearing mucus. Examples of such conditions include cystic fibrosis and Primary Ciliary Dyskinesia (PCD). When only a portion of the lung is affected, the disorder may be caused by an obstruction rather than a medical condition.

The methods described herein for treating or preventing (i.e., reducing the likelihood of occurrence of) an aging-related lung disease or disorder can also be used to treat a subject that is aging and has loss (or regression) of lung function (i.e., reduced or impaired lung function compared to a younger subject) and/or degeneration of lung tissue. The respiratory system undergoes various anatomical, physiological and immunological changes with age. This structural change includes chest wall and thoracic spine deformations, which can compromise the overall respiratory compliance, resulting in more effort to breathe. The respiratory system undergoes structural, physiological and immunological changes with age. An increased proportion of neutrophils and a lower percentage of macrophages can be found in bronchoalveolar lavage (BAL) fluid of older adults compared to younger adults. Sustained low-grade inflammation of the lower respiratory tract can cause proteolytic and oxidant-mediated damage to the lung matrix, leading to loss of alveolar cells and impaired gas exchange across the alveolar membranes observed with aging. Persistent inflammation of the lower respiratory tract may predispose the elderly to increased susceptibility to toxic environmental exposure, as well as accelerated decline in lung function. (see, e.g., Sharma et al, Clinical Intercations in Aging 1:253-60 (2006)). In aging, oxidative stress exacerbates inflammation (see, e.g., Brod, Inflamm Res 2000; 49: 561-. In aging, alterations in redox balance and increases in oxidative stress contribute to cytokine, chemokine and adhesion molecule, and enzyme expression (see, e.g., Chung et al, Ageing Res Rev 2009; 8: 18-30). Constitutive activation and recruitment of macrophage cells, T cells, and mast cells promotes the release of proteases that lead to extracellular matrix degradation, cell death, remodeling, and other events, which can lead to tissue and organ damage during chronic inflammation (see, e.g., Demedts et al, Respir Res 2006; 7: 53-63). By administering senolytic agents to an aging subject, including asymptomatic middle-aged people, the decline in lung function can be slowed or inhibited by killing and removing senescent cells from the respiratory tract.

The effectiveness of a senolytic agent can be readily determined by one of skill in the medical and clinical arts. One or any combination of diagnostic methods, including physical examination, evaluation and monitoring of clinical symptoms, and performance of the analytical tests and methods described herein, may be used to monitor the health status of a subject. The effect of treatment with senolytic agents or pharmaceutical compositions comprising the agents can be analyzed using techniques known in the art, such as comparing the symptoms of a patient suffering from or at risk of a pulmonary disease who has received treatment with the symptoms of a patient who has not received such treatment or who has received placebo treatment. Furthermore, an assessment of the mechanical functionality of the lungs can be carried outMethods and techniques, such as techniques for measuring lung volume, elasticity, and airway hypersensitivity. To determine lung function and monitor lung function throughout treatment, any of a variety of metrics may be obtained, the amount of complementary Exhalation (ERV), Forced Vital Capacity (FVC), Forced Expiratory Volume (FEV) (e.g., FEV in one second, FEV1), FEV1/FEV ratio, forced expiratory flow of 25% to 75%, and Maximum Voluntary Ventilation (MVV), maximum expiratory flow (PEF), Slow Vital Capacity (SVC). Total lung volume includes total lung volume (TLC), Vital Capacity (VC), Residual Volume (RV), and functional residual volume (FRC). Carbon monoxide Diffusivity (DLCO) can be used to measure gas exchange across the alveolar-capillary membrane. Peripheral capillary blood oxygen saturation (SpO) can also be measured₂) (ii) a Normal oxygen levels are typically between 95% and 100%. SpO of less than 90%₂The level indicates that the subject has hypoxemia. Values below 80% are considered dangerous and require intervention to maintain brain and heart function and avoid cardiac or respiratory arrest.

Neurological diseases and disorders.Senescence-associated diseases or disorders that can be treated by administration of a senolytic agent described herein include neurological diseases or disorders. Such age-related diseases and disorders include parkinson's disease, alzheimer's disease (and other dementias), Motor Neuron Dysfunction (MND), Mild Cognitive Impairment (MCI), huntington's disease, and diseases and disorders of the eye such as age-related macular degeneration. Other eye diseases associated with increased age are glaucoma, hypopsia, presbyopia and cataracts.

Parkinson's Disease (PD) is the second most common neurodegenerative disease. It is a state of disability of the brain characterized by slow movement (bradykinesia), shaking, stiffness, and loss of balance in the late stages. Many of these symptoms are due to the loss of certain nerves in the brain, which results in dopamine deficiency. This disease is characterized by neurodegeneration, such as loss of dopaminergic neurons in approximately 50% to 70% of the substantia nigra pars compacta, extensive loss of dopamine in the striatum, and/or the presence of intracytoplasmic inclusions (lewisite) composed mainly of alpha-synuclein and ubiquitin. Parkinson's disease is also characterized by motor deficits, such as tremor, rigidity, bradykinesia, and/or postural instability. Subjects at risk of developing parkinson's disease include those with a family history of parkinson's disease and those exposed to pesticides (e.g., rotenone or paraquat), herbicides (e.g., orange), or heavy metals. Aging of dopamine-producing neurons is thought to result in cell death observed in PD through the production of reactive oxygen species (see, e.g., Cohen et al, j.neural frans.suppl.19: 89-103 (1983)); thus, the methods and senolytic agents described herein are useful for the treatment and prevention of parkinson's disease.

Methods for detecting, monitoring or quantifying neurodegenerative and/or motor deficits associated with parkinson's disease are known in the art, such as histological studies, biochemical studies, and behavioral assessments (see, e.g., U.S. application publication No. 2012/0005765). Symptoms of parkinson's disease are known in the art and include, but are not limited to, starting or ending voluntary motor difficulties, twitching, movement stiffness, muscle atrophy, shaking (tremor), and changes in heart rate but normal reflexes, bradykinesias, and postural instability. It is increasingly recognized that people diagnosed with parkinson's disease may have cognitive disorders, including mild cognitive disorders, in addition to their physical symptoms.

Alzheimer's Disease (AD) is a neurodegenerative disease that exhibits a slowly progressive intellectual decline with memory impairment, disorientation and confusion, resulting in severe dementia. Age is the only largest risk factor for the development of AD, which is the leading cause of dementia in elderly (see, e.g., Hebert, et al, arch. neuron.60: 1119-1122 (2003)). Early clinical symptoms show significant similarity to mild cognitive impairment (see below). As the disease progresses, disorientation, confusion, behavioral changes, disorientation, and difficulty in walking and swallowing arise.

Alzheimer's disease is characterized by the presence of neurofibrillary tangles and amyloid (senescent) plaques in histological samples. The disease mainly affects the limbic and cortical areas of the brain. The argentophilic plaques, which contain amyloid-forming a β fragments of the amyloid-like precursor protein (APP), are spread throughout the cerebral cortex and hippocampus. Neurofibrillary tangles are found in pyramidal neurons located primarily in the neocortex, hippocampus, and the basal nucleus of Meynert (Meynert). Other changes were observed, such as degeneration of particulate vacuoles in pyramidal cells of the hippocampus, and neuronal loss and gliosis in the cortex and hippocampus. Subjects at risk of developing alzheimer's disease include those of high age, those with a family history of alzheimer's disease, those with a genetic risk gene (e.g., ApoE4) or definitive gene mutation (e.g., APP, PS1, or PS2), and those with a history of head trauma or cardiac/vascular conditions (e.g., hypertension, heart disease, stroke, diabetes, high cholesterol).

Some behavioral and histopathological tests for assessing the alzheimer's disease phenotype, for characterizing therapeutic agents, and evaluating treatment are known in the art. Histological analysis is usually performed post mortem. Histological analysis of a β levels can be performed using thioflavin-S. Congo Red, or anti-A.beta.staining (e.g., 4G8, 10D5, or 6E10 antibodies) visualizes A.beta.deposits on sliced brain tissue (see, e.g., Holcomb et al, 1998, Nat. Med.4: 97-100; Borchelt et al, 1997, Neuron19: 939-. In vivo methods for visualizing Α β deposits in transgenic mice have also been described. BSB ((trans, trans) -1-bromo-2, 5-bis- (3-hydroxycarbonyl-4-hydroxy) styrylbenzene) and PET tracer¹¹C-labeled Pittsburgh compound-B (PIB) binds to A β plaques (see, e.g., Skovronsky et al, 2000, Proc. Natl. Acad. Sci. USA 97: 7609-7614; Klunk et al, 2004, Ann. neurol.55: 306-319). Comprises¹⁹The amyloidophilic congo red-type compound of F, FSB ((E, E) -1-fluoro-2, 5-bis- (3-hydroxycarbonyl-4-hydroxy) styrylbenzene), allows visualization of A β plaques by MRI (see, e.g., Higuchi et al, 2005, Nature Neurosci.8: 527-. Radiolabeled, putrescine-modified amyloid- β peptide labels amyloid deposits in a mouse model of Alzheimer's disease in vivo (see, e.g., Wengenack et al, 2000, nat. Biotechnol.18:868 872).

Glial Fibrillary Acidic Protein (GFAP), increased by astrocytes, is a marker of astrocyte activation and gliosis during neurodegeneration. A β plaques are associated with GFAP-positive activated astrocytic fine and can be visualized by GFAP staining (see, e.g., Nagele et al, 2004, Neurobiol. aging 25: 663-. Neurofibrillary tangles can be identified by immunohistochemistry using thioflavin-S fluorescence microscopy and Gallyas silver staining (see, e.g., Gotz et al, 2001, J.biol.chem.276: 529-. Axonal transport and axonal staining studies by electron microscopy are useful for neuronal degeneration (see, e.g., Ishihara et al, 1999, Neuron24: 751-762).

Standard diagnostic methods known in the art for alzheimer's disease can be used to identify subjects suffering from alzheimer's disease. Generally, the diagnosis of alzheimer's disease is based on the patient's symptoms (e.g., progressive decline in memory function, progressive abandonment and frustration of normal activities, apathy, restlessness or irritability, aggression, anxiety, sleep disorders, vexation, abnormal motor behavior, disinhibition, social withdrawal, decreased appetite, hallucinations, dementia), medical history, neuropsychological tests, neurological and/or physical examinations. Various proteins associated with Alzheimer's pathology, including tau, amyloid beta peptide, and AD7C-NTP, can also be tested in cerebrospinal fluid. Gene testing can also be used for early-onset familial Alzheimer's disease (eFAD), autosomal dominant hereditary diseases. Clinical gene testing can be used for individuals with symptoms of AD or at risk family members of patients with early onset disease. In the united states, PS2 mutations and APP can be tested in clinical or federally approved clinical laboratories according to the clinical laboratory improvement amendments. Commercial testing of the PS1 mutation is also available (Elan Pharmaceuticals).

One skilled in the medical and clinical arts can readily determine the effectiveness of one or more senolytic agents described herein and monitor a subject receiving one or more senolytic agents. One or any combination of diagnostic methods, including physical examination, evaluation and monitoring of clinical symptoms, and performance of the analytical tests and methods described herein, can be used to monitor the health status of a subject. The effect of administration of one or more senolytic agents can be analyzed using techniques known in the art, such as comparing the symptoms of a patient who has received treatment, or is at risk of alzheimer's disease, to the symptoms of a patient who has not received such treatment, or received placebo treatment.

Mild Cognitive Impairment (MCI). MCI is a brain function syndrome that involves the onset and progression of cognitive disorders beyond that expected according to an individual's age and education, but is not significant enough to interfere with that individual's daily activities. MCI is an aspect of cognitive aging that is considered to be a transitional state between normal aging and the dementia to which it may be transformed (see Pepeu, diagnostics in Clinical Neuroscience 6:369-377, 2004). MCIs that primarily affect memory are referred to as "amnestic MCIs". Patients with amnestic MCI may begin to forget important information that they would have easily remembered before, such as recent events. Amnestic MCI is common in the prodromal phase of alzheimer's disease. MCI that affect thinking skills rather than memory are called "non-amnestic MCI". This type of MCI affects thinking skills such as the ability to make correct decisions, determine the order or timing of steps required to complete complex tasks, or know sight. It is believed that individuals with non-amnestic MCI are more likely to transition to other types of dementia (e.g., dementia with lewy bodies).

Persons in the medical field are increasingly aware that persons diagnosed with parkinson's disease may have MCI in addition to their physical symptoms. Recent studies have shown that 20-30% of people with parkinson's disease have MCI, and that their MCI tends to be non-amnesic. Parkinson's disease patients with MCI will sometimes progress further to global dementia (Parkinson's disease with dementia).

Methods for detecting, monitoring, quantifying, or assessing the neuropathological deficits associated with MCI are known in the art, and include astrocytic morphometry, acetylcholine release, silver staining for assessing neurodegeneration, and PiB PET imaging for detecting beta amyloid deposits (see, e.g., U.S. application publication No. 2012/0071468; Pepeu,2004, supra). Methods for detecting, monitoring, quantifying, or evaluating behavioral deficits associated with MCI are also known in the art, including eight sets of radial maze paradigms, non-sample matching tasks, non-self-location determination tasks in water maze, morris maze testing, visuospatial tasks and delayed response spatial memory tasks, olfactory novelty tests (see, supra).

Motor Neuron Dysfunction (MND). MNDs are a class of progressive neurological disorders that destroy motor neurons, i.e., cells that control essential voluntary muscle activity such as speaking, walking, breathing, and swallowing. Degeneration is classified according to whether it affects the upper motor neurons, the lower motor neurons, or both. Examples of MNDs include, but are not limited to, Amyotrophic Lateral Sclerosis (ALS), also known as lugal leid Disease (Lou Gehrig's Disease), progressive bulbar palsy, pseudobulbar palsy, primary lateral sclerosis, progressive muscular atrophy, lower motor neuron Disease, and Spinal Muscular Atrophy (SMA) (e.g., SMA1, also known as Werdnig-Hoffmann Disease, SMA2, SMA3, also known as Kugelberg-Welander Disease and Kennedy's Disease), post-polio syndrome, and hereditary spastic paraplegia. In adults, the most common MND is Amyotrophic Lateral Sclerosis (ALS), which affects both upper and lower motor neurons. Which may affect arm, leg or facial muscles. Primary lateral sclerosis is a disease of upper motor neurons, while progressive muscular atrophy affects only lower motor neurons in the spinal cord. In progressive bulbar palsy, the lowermost motor neurons of the brain stem are most affected, leading to slurred speech and difficulty chewing and swallowing. There are almost always signs of mild abnormalities in the arms and legs. MND patients exhibit a phenotype of parkinson's disease (e.g., with tremor, rigidity, bradykinesia, and/or postural instability). Methods for detecting, monitoring, or quantifying motor and/or other defects associated with parkinson's disease, such as MND, are known in the art (see, e.g., U.S. application publication No. 20120005765).

Methods for detecting, monitoring, quantifying, or evaluating motor and histopathological defects associated with MNDs are also known in the art, including histopathological, biochemical, and electrophysiological studies as well as motor activity analysis (see, e.g., Rich et al, J Neurophysiol 88: 3293-. Histopathologically, MNDs are characterized by death of motor neurons, progressive accumulation of detergent-resistant aggregates containing SOD1 and ubiquitin, and abnormal neurofilament accumulation in degenerative motor neurons. In addition, reactive astroglia and microglia are often detected in diseased tissues. MND patients exhibit one or more motor deficits including muscle weakness and atrophy, uncontrollable convulsions, spasms, slow motor exertion, and hyperactive tendinous reflexes.

Ophthalmic diseases and disorders: in certain embodiments, the aging-related disease or disorder is an ocular disease, disorder, or condition, e.g., presbyopia, macular degeneration, or cataract. In certain other embodiments, the disease or disorder associated with aging is glaucoma. Macular degeneration is a neurodegenerative disease that results in the loss of photoreceptor cells in the central portion of the retina, known as the macula. Macular degeneration is generally classified into two categories: dry type and wet type. The dry form is more common than the wet form, and about 90% of age-related macular degeneration (ARMD or AMD) patients are diagnosed with the dry form. The wet form of the disease usually results in more severe vision loss. While the exact cause of age-related macular degeneration is still unknown, the number of aging Retinal Pigment Epithelial (RPE) cells increases with age. Age and certain genetic and environmental factors are risk factors for developing ARMD (see, e.g., Lyengar et al, am.j.hum.gene. 74:20-39 (2004)) (Epub 2003December 19); Kenealy et al, mol.vis.10:57-61 (2004); Gorin et al, mol.vis.5:29 (1999)). Environmental pathogenesis includes omega-3 fatty acid uptake (see, e.g., Christen et al, Arch Ophthalmol. 129:921-29 (2011)); estrogen exposure (see, e.g., Feshanich et al, Arch Ophthalmol 126(4):519-24) (2008)); and increased serum levels of vitamin D (see, e.g., Millen, et al, Arch Ophthalmol.129(4):481-89 (2011)). Genetically induced risk factors include reduced levels of Dicer1 (an enzyme associated with microrna maturation) in the eyes of dry AMD patients, with reduced micrornas contributing to the senescent cell profile; and DICER1 ablation causes premature aging (see, e.g., mudharani j. cell. biol. (2008)).

Dry ARMD is associated with atrophy of the RPE layer, which leads to loss of photoreceptor cells. Dry ARMD may result from aging and thinning of macular tissue and pigmentation in the macula. Aging appears to inhibit the replication and migration of RPE, resulting in permanent depletion of RPE in the macula of patients with dry AMD (see, e.g., Iriyama et al, j.biol. chem.283: 11947-. For wet ARMD, new blood vessels grow under the retina and leak blood and fluid. This abnormally leaky choroidal neovascularization leads to retinal cell death, producing a central blindness point. Different forms of macular degeneration may also occur in younger patients. Non-age-related causes may be associated with genetics, diabetes, malnutrition, head injury, infection, or other factors.

The loss of vision noted by the patient or by the ophthalmologist during routine eye examination may be the primary indicator of macular degeneration. Formation of bruch's subretinal exudate of the macula, or "drusen," has historically been the first sign of possible progression of macular degeneration. Symptoms include straight line perceptual distortion, and in some cases, the center of the field of view appears to be more distorted than the rest of the scene; dark, blurred regions or "whiteout" occur in the center of the field of view; and/or a change or impairment of color perception. Diagnosis and monitoring of subjects with macular degeneration can be accomplished by one skilled in the art of ophthalmology according to art recognized periodic eye examination procedures and reports of symptoms by the subject.

Presbyopia is an age-related condition in which the eye exhibits a progressively reduced ability to focus on near objects as the speed and magnitude of adaptation of the normal eye decreases with age. Lens elasticity loss and ciliary muscle contraction loss have been postulated as their causes (see, e.g., Heys et al, 2004, mol. Vis.10: 956-63; Petrash,2013, invest. Ophthalmol. Vis. Sci. 54: ORSF54-ORSF 59). Age-related changes in the mechanical properties of the anterior and posterior lens capsules suggest that the mechanical strength of the posterior lens capsule decreases significantly with age (see, e.g., Krag et al, invest. Ophthalmol. Vis. Sci.44:691-96 (2003); Krag et al, invest. Ophthalmol. Vis. Sci.38:357-63 (1997)).

The layered structure of the pouch also changes and is caused, at least in part, by changes in the composition of the tissue (see, e.g., Krag et al, 1997, supra, and references cited therein). The major structural component of the lens capsule is basement membrane type IV collagen organized into a three-dimensional molecular network (see, e.g., Cummings et al, connect. tissue Res.55:8-12 (2014); Veis et al, Coll. Relat. Res.1981; 1: 269-86). Type IV collagen consists of 6 homologous alpha chains (. alpha.1-6) associated as heterotrimeric IV collagenases, each of which comprises a specific chain combination of alpha 112, alpha 345 or alpha 556 (see, e.g., Khoshenoodi et al, Microsc. Res. Tech.2008; 71: 357-70). The tripeptide sequences of protomer and Gly-X-Y share the structural similarity of the triple-helical collagen domain (Timpl et al, Eur. J. biochem. 1979; 95: 255-263) and end up in a globular C-terminal region called the non-collagenous 1(NC1) domain. The N-terminus consists of a helical domain called the 7S domain (see, e.g., Ristelli et al, Eur. J. biochem. 1980; 108: 239-250), which is also involved in the protomer-protomer interaction.

Studies have revealed that IV collagen affects cellular function, which is inferred from the localization of basement membrane under the epithelial layer and data supporting the role of IV collagen in tissue stabilization (see, e.g., Cummings et al, supra). Posterior Capsular Opacification (PCO) will develop as a complication in about 20-40% of patients in the years following cataract surgery (see, e.g., Awasthi et al, Arch Ophthalmol.2009; 127: 555-62). PCO results from the proliferation and activity of residual lens epithelial cells along the posterior capsule in a response similar to wound healing (see, e.g., Awasthi et al, archophthalmol.2009; 127: 555-62). Growth factors such as fibroblast growth factor, transforming growth factor beta, epidermal growth factor, hepatocyte growth factor, insulin-like growth factor, and interleukins IL-1 and IL-6 may also promote epithelial cell migration (see, e.g., Awasthi et al, supra; Raj et al, supra). As discussed above, these factors and cytokines produced by senescent cells contribute to SASP. In contrast, in vitro studies have shown that IV collagen promotes the adhesion of lens epithelial cells (see, e.g., Olivero et al, invest. Ophthalmol. Vis. Sci.1993; 34: 2825-34). Adhesion of IV collagen, fibronectin, and laminin to the intraocular lens inhibits cell migration and may reduce the risk of PCO (see, e.g., Raj et al, int.J.biomed.Sci.2007; 3: 237-50).

Without wishing to be bound by any particular theory, selective killing of senescent cells by the senolytic agents described herein may slow or hinder (delay, inhibit, delay) the structural breakdown of the type IV collagen network (disassociation). Removal of senescent cells, and thus the inflammatory effects of SASP, can reduce or inhibit epithelial cell migration, and can also delay (inhibit) the onset of presbyopia, or reduce or slow the progressive exacerbation of the condition (e.g., slow progression from moderate to mild, or from severe to moderate). The senolytic agents described herein may also be used after cataract surgery to reduce the likelihood of PCO recurrence.

Although no direct evidence was obtained from human studies that cellular senescence is associated with cataract development, the BubR1 suballent allele mouse develops bilateral posterior subcapsular cataracts early in life, suggesting that senescence may play a role (see, e.g., Baker et al, nat. cell biol. 10: 825-36 (2008)). Cataracts are clouding of the lens of the eye, resulting in blurred vision and, if left untreated, may result in blindness. Surgery is effective and is performed periodically to remove cataracts. Administration of one or more senolytic agents described herein can result in a reduction in the likelihood of cataract recurrence, or can slow or inhibit the progression of cataract. The presence and severity of cataracts can be detected by eye examination by one skilled in the ophthalmic art using conventional methods.

In certain embodiments, at least one senolytic agent that selectively kills senescent cells can be administered to a subject at risk of developing presbyopia, cataract, or macular degeneration. Treatment with senolytic agents may be initiated when a human subject is at least 40 years of age to delay or inhibit the onset or progression of cataracts, presbyopia, and macular degeneration. Because nearly all people develop presbyopia, in certain embodiments, a senolytic agent can be administered to a human subject after the subject reaches 40 years of age in a method as described herein to slow or inhibit the onset or progression of presbyopia.

In certain embodiments, the aging-related disease or disorder is glaucoma. Glaucoma is a broad term used to describe a group of diseases that cause visual field loss, often without any other obvious symptoms. The lack of symptoms often results in delayed diagnosis of glaucoma until the end of the disease. Even subjects with glaucoma do not become blind, but their vision is often severely impaired. Typically, the transparent fluid flows into and out of the anterior segment of the eye, known as the anterior chamber. In individuals with wide angle glaucoma, this fluid drains too slowly, resulting in increased intraocular pressure. If left untreated, this high pressure then damages the optic nerve and may lead to complete blindness. Loss of peripheral vision is caused by the death of ganglion cells in the retina. Ganglion cells are a special type of projection neurons that connect the eye to the brain. A four-fold increase in aging was observed in glaucoma patients when the cellular network required for fluid outflow was stained with SA- β -Gal (see, e.g., Liton et al, Exp. Gerontol.40: 745-748 (2005)).

To monitor the effect of therapy on inhibiting the progression of glaucoma, the most widely used technique is standard automated visual field measurements (visual field examination). In addition, several algorithms for progress monitoring have been developed (see, e.g., Wesselink et al, Arch Ophthalmol.127(3):270- & 274 (2009), and references therein). Other methods include gonioscopy (examining the angle at which the trabecular meshwork and fluid drain out of the eye); imaging techniques such as laser tomography (e.g., HRT3), laser polarimetry (e.g., GDX), and ocular coherence tomography; examining the eye; and pachymetric measurements to determine the central thickness of the cornea.

A metabolic disease or disorder.Senescence-associated diseases or disorders that can be treated by administration of a senolytic agent include metabolic diseases or disorders. Such diseases and disorders associated with aging cells include diabetes, metabolic syndrome, diabetic ulcers and obesity.

Diabetes is characterized by high levels of blood glucose due to defects in insulin production, insulin action, or both. The vast majority (90 to 95%) of adults with diabetes are diagnosed with type 2 diabetes, which is characterized by a gradual loss of insulin production by the pancreas. Diabetes is the leading cause of kidney failure, non-traumatic lower limb amputation, and new cases of blindness in adults in the united states. Diabetes is a significant cause of heart Disease and stroke, and is the seventh leading cause of death in the United States (see, e.g., Centers for Disease Control and prediction, National Diabetes mellitus panels: National Diabetes and genetic information on Diabetes and pre-Diabetes the United States,2011 ("Diabetes face"). The senolytic agents described herein are useful for treating type 2 diabetes, particularly age, diet, and obesity-related type 2 diabetes.

The involvement of senescent cells as responses to injury or metabolic dysfunction in metabolic diseases such as obesity and type 2 diabetes has been revealed (see, e.g., Tchkonia et al, Aging Cell 9:667-684 (2010)). Adipose tissue from obese mice showed induction of the aging markers SA-. beta. -Gal, p53, and p21 (see, e.g., Tchkonia et al, supra; Minamino et al, nat. Med.15: 1082-1087 (2009)). Concomitant upregulation of pro-inflammatory cytokines such as tumor necrosis factor-alpha and Ccl2/MCP1 was observed in the same adipose tissue (see, e.g., Minamino et al, supra). The induction of senescent cells in obesity may be of clinical significance because it is also suggested that the pro-inflammatory SASP component contributes to type 2 diabetes (see, e.g., Tchkonia et al, supra). Upregulation of similar patterns of aging markers and SASP components has been associated with diabetes in mice and humans (see, e.g., Minamino et al, supra). Thus, the methods described herein comprising administering an aging scavenger may be useful for the treatment or prevention of type 2 diabetes as well as obesity and metabolic syndrome. Without wishing to be bound by theory, killing senescent preadipocytes by contacting them with a senolytic agent may provide clinical and health benefits to a person having any one of diabetes, obesity, or metabolic syndrome.

Subjects suffering from type 2 diabetes can be identified using standard diagnostic methods known in the art for type 2 diabetes. Generally, diagnosis of type 2 diabetes is based on the patient's symptoms (e.g., thirst and increased frequency of urination, increased hunger, weight loss, weakness, blurred vision, slow wound healing or repeated infection, and/or areas of darkened skin), medical history, and/or physical examination. Subjects at risk of developing type 2 diabetes include persons with a family history of type 2 diabetes, as well as persons with other risk factors such as overweight, body fat distribution, inactivity, race, age, prediabetes, and/or gestational diabetes.

The effectiveness of a senolytic agent can be readily determined by one of skill in the medical and clinical arts. One or any combination of diagnostic methods, including physical examination, evaluation and monitoring of clinical symptoms, and performance of analytical tests and methods such as those described herein can be used to monitor the health status of a subject. For example, a subject receiving one or more senolytic agents described herein to treat or prevent diabetes is monitored by determining glucose and insulin resistance, energy expenditure, body composition, adipose tissue, skeletal muscle, and liver inflammation, and/or lipotoxicity (muscle and liver lipids determined by in vivo imaging and muscle, liver, bone marrow, and pancreatic beta cell lipid accumulation and inflammation determined histologically). Other characteristic features or phenotypes of type 2 diabetes are known and can be determined as described herein and by using other methods and techniques known and routinely applied in the art.

Obesity and obesity-related disorders can be used to refer to conditions in a subject whose body weight measurement is greater than ideal for its height and build (frame). Body Mass Index (BMI) is a measure for determining overweight, and is calculated from the height and weight of a subject. A person is considered overweight when it has a BMI of 25-29; a human is considered obese when it has a BMI of 30-39; while a person is considered to be severely obese when they have a BMI of ≧ 40. Thus, the terms obesity and obesity related refer to human subjects having a body mass index value of more than 30, more than 35, or more than 40. One class of obesity not summarized by BMI is known in the art as "abdominal obesity," which involves the discovery of additional fat near the waist of a subject, which is an important factor in health even though it is not related to BMI. The simplest and most common measure of abdominal obesity is waist circumference. Abdominal obesity is generally defined as waist circumference of 35 inches or more in women and 40 inches or more in men. More sophisticated methods for determining obesity require special equipment such as magnetic resonance imaging or dual energy X-ray absorption measurement machines.

One condition or disorder associated with diabetes and aging is diabetic ulcers (i.e., diabetic wounds). Ulcers are breaks in the skin that may extend to involve subcutaneous tissue or even muscle or bone. These injuries occur particularly in the lower extremities. Patients with diabetic venous ulcers exhibit an increased presence of cellular senescence at the site of the chronic wound (see, e.g., Stanley et al, (2001) J.Vas.Surg.33: 1206-1211). Chronic inflammation is also observed at chronic wound sites, such as diabetic ulcers (see, e.g., Goren et al, (2006) am.j.pathol. 7168: 65-77; Seitz et al, (2010) exp.diabetes res.2010:476969), suggesting that the pro-inflammatory cytokine phenotype of aging cells has an effect on pathology.

A subject with type 2 diabetes or at risk of developing type 2 diabetes may have metabolic syndrome. The metabolic syndrome in humans is often associated with obesity and is characterized by one or more of cardiovascular disease, hepatic steatosis, hyperlipidemia, diabetes and insulin resistance. Subjects with metabolic syndrome may present with a range of metabolic disorders or abnormalities, which may include, for example, hypertension, type 2 diabetes, hyperlipidemia, dyslipidemia (e.g., hypertriglyceridemia, hypercholesterolemia), insulin resistance, hepatic steatosis (steatohepatitis), hypertension, atherosclerosis, and other metabolic disorders.

Renal insufficiency: nephrological conditions such as glomerulopathy occur in the elderly. Glomerulonephritis is characterized by inflammation of the kidney and expression of two proteins, IL1 α and IL1 β (see, e.g., Niemir et al, (1997) Kidney Int.52: 393-403). IL1 α and IL1 β are considered to be the primary regulators of SASP (see, e.g., Coppe et al (2008) plos. biol.6: 2853-68). Glomerular disease is associated with an increase in the presence of senescent cells, particularly in fibrotic kidneys (see, e.g., Sis et al, (2007) Kidney int 71: 218-226).

A skin disease or disorder.Age-related diseases or disorders that can be treated by administration of the senolytic agents described herein include skin diseases or disorders. Such diseases and disorders associated with aging cells include psoriasis and eczema, which are also inflammatory diseases and are discussed in more detail above. Other skin diseases and conditions associated with aging include wrinkles (wrinkles due to aging); pruritus (associated with diabetes and aging); dysesthesia (chemotherapy side effects associated with diabetes and multiple sclerosis); psoriasis (as described above) and other papulosquamous disorders, e.g., erythroderma, lichen planus, and lichenification; atopic dermatitis (a form of eczema and associated with inflammation); eczematoid skin rashes (often observed in elderly patients and associated with side effects of certain drugs). Other skin diseases and conditions associated with aging include eosinophilic skin disease (associated with certain types of hematological cancers); reactive neutrophilic skin disorders (associated with underlying diseases such as inflammatory bowel syndrome); pemphigus (autoimmune disease in which autoantibodies are formed against desmoglein); pemphigoid and other immune bullous skin diseases (autoimmune blistering of the skin); fibrohistiocyte proliferation of the skin, which is associated with aging; and cutaneous lymphomas more common in the elderly population. Other skin diseases that can be treated according to the methods described herein include cutaneous lupus, which is a symptom of lupus erythematosus. Late onset lupus may be associated with decreased T cell and B cell function (i.e., decline) and cytokines (immunosenescence) associated with aging.

Transfer of. In particular embodiments, methods are provided for treating or preventing (i.e., reducing the likelihood of occurrence or development of) a disease (or disorder or condition) associated with senescent cells, i.e., metastasis. And alsoSenolytic agents described herein can be used to treat or prevent (i.e., reduce the likelihood of occurrence or development) metastasis (i.e., spread and spread of cancer or tumor cells) of one organ or tissue to another in vivo according to the methods described herein.

The senescent cell-associated disease or disorder includes metastasis, and a subject with cancer may benefit from administration of a senolytic agent for inhibiting metastasis as described herein. Such senolytic agents can inhibit tumor proliferation when administered to a subject having cancer according to the methods described herein. Cancer metastasis occurs when cancer cells (i.e., tumor cells) spread beyond the original anatomical location and begin to metastasize to other areas throughout the body of a subject. Tumor proliferation can be determined by tumor size, which can be determined in a variety of ways familiar to those skilled in the art, such as, for example, by PET scan, MRI, CAT scan, biopsy. The effect of a therapeutic agent on tumor proliferation can also be assessed by examining the differentiation of tumor cells.

As used herein and in the art, the term cancer or tumor is a clinically descriptive term that includes diseases generally characterized by cells exhibiting abnormal cell proliferation. The term cancer is commonly used to describe malignant tumors or disease states resulting from tumors. Alternatively, abnormal growth may be referred to in the art as neoplasms. As with reference to tissue, the term tumor generally refers to any abnormal tissue growth characterized at least in part by excess and abnormal cell proliferation. The tumor may be metastatic and able to spread beyond its original anatomical location and begin to colonize other areas within the subject's entire body. The cancer may comprise a solid tumor or may comprise a "liquid" tumor (e.g., leukemia and other hematologic cancers).

Cells are induced to age by cancer therapies such as radiation and certain chemotherapeutic drugs. The presence of senescent cells increases the secretion of inflammatory molecules (see the description of senescent cells herein), promotes tumor progression, which may include promoting tumor growth and increasing tumor size, promoting metastasis, and altering differentiation. When aging cells are destroyed, tumor progression is significantly inhibited, resulting in a small tumor size and no or less metastatic growth is observed (see, e.g., international application publication No. WO 2013/090645).

In one embodiment, a method for preventing (i.e., reducing the likelihood of occurrence), inhibiting, or delaying metastasis in a subject having cancer by administering a senolytic agent as described herein is provided. In particular embodiments, the senolytic agent is administered on one or more days within a treatment window (i.e., course) of no more than 7 days or 14 days. In other embodiments, the course of treatment is no longer than 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or no longer than 21 days. In other embodiments, the course of treatment is one day. In certain embodiments, the senolytic agent is administered on two or more days within a treatment window of no more than 7 days or 14 days, on 3 or more days within a treatment window of no more than 7 days or 14 days; 4 or more days within a treatment window of no more than 7 or 14 days; (ii) administration for 5 or more days within a treatment window of no more than 7 or 14 days; administration is on days 6, 7, 8, 9, 10, 11, 12, 13, or 14 within a treatment window of no more than 7 or 14 days. In certain embodiments, when at least one senolytic agent is administered to a subject for a treatment window of 3 days or longer, the agent may be administered every 2 days (i.e., every other day). In certain other embodiments, when at least one senolytic agent is administered to a subject for a treatment window of 4 days or longer, the agent can be administered every 3 days (i.e., every third day).

Because cells are senescent induced by cancer therapies such as radiation and certain chemotherapeutic drugs (e.g., doxorubicin; paclitaxel; gemcitabine; pomalidomide; lenalidomide), the senolytic agents described herein may be used after the chemotherapy or radiation therapy to kill (or promote the killing of) the senescent cells. As discussed herein and understood in the art, establishment of senescence (as shown by the presence of a senescence-associated secretory phenotype (SASP)) occurs over days; thus, senolytic agents are administered to kill senescent cells when senescence is established, and thereby reduce the likelihood of occurrence or reduce the extent of metastasis. As discussed herein, the following course of treatment for administration of senolytic agents can be used in a method for treating or preventing (i.e., reducing the likelihood of occurrence or reducing the severity of) chemotherapy or radiotherapy side effects as described herein.

In certain embodiments, when the chemotherapy or radiotherapy is under treatment (i.e., chemotherapy or radiotherapy) for at least one day followed by a treatment cycle of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 (or about 2 weeks), 15, 16, 17, 18, 19, 20, 21 days (or about 3 weeks), or about 4 weeks (about one month) of non-treatment (i.e., non-chemotherapy or radiotherapy), the senolytic agent is administered on one or more days during a non-treatment time interval (time period), wherein the one or more days begin on or after the second day of the non-treatment time interval and end on or before the last day of the non-treatment time interval. By way of illustrative example, if n is a non-treatment day, the senolytic agent is administered on at least one day and no more than n-1 days of the non-treatment time interval. In certain particular embodiments, when the chemotherapy or radiation therapy is under treatment (i.e., chemotherapy or radiation therapy) for at least one day followed by at least one week of non-treatment cycles, the senolytic agent is administered on one or more days during the non-treatment time interval, wherein the one or more days begin on or after the second day of the non-treatment time interval and end on or before the last day of the non-treatment time interval. In a more specific embodiment, when chemotherapy or radiation therapy is administered for at least one day under treatment (i.e., chemotherapy or radiation therapy) followed by a non-therapeutic treatment cycle for at least one week, the senolytic agent is administered on day six of the non-therapeutic time interval. In other specific embodiments, when the chemotherapy or radiation therapy is under treatment (i.e., chemotherapy or radiation therapy) for at least one day followed by a treatment period of at least two weeks other than treatment, the senolytic agent is administered beginning on the sixth day of the non-chemotherapy or non-radiation therapy time interval and ending at least one or at least two days prior to the first day of the subsequent chemotherapy or radiation therapy session. By way of example, if the non-chemotherapy or non-radiotherapy time interval is two weeks, the senolytic agent can be administered beginning on the sixth day after the end of the chemotherapy or radiotherapy session (i.e., the sixth day of the non-chemotherapy/radiotherapy interval) and at least one day and no more than 7 days of the non-treatment time interval (i.e., 1, 2,3, 4, 5, 6, or 7 days). When the non-chemotherapeutic or non-radiotherapeutic interval is at least three weeks, the senolytic agent can be administered at least one day and no more than 14 days (i.e., 1-14 days: 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days) of the non-therapeutic interval, beginning on the sixth day after the end of the chemotherapeutic or radiotherapeutic session. In other embodiments, the senolytic agent course of treatment is at least one day and no longer than 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days or no more than 21 days (i.e., 1-21 days) depending on the interval without chemotherapy or radiotherapy, provided that administration of the senolytic agent does not occur simultaneously with chemotherapy or radiotherapy. In certain embodiments, the senolytic agent course of treatment is one day. In certain embodiments, the senolytic agent is administered on two or more days within a treatment window of no more than 14 days, on 3 or more days within a treatment window of no more than 14 days; (ii) administration for 4 or more days within a treatment window of no more than 14 days; (ii) administration for 5 or more days within a treatment window of no more than 14 days; administered on days 6, 7, 8, 9, 10, 11, 12, 13, or 14 within a treatment window of no more than 14 days. In certain embodiments, when the at least one senolytic agent is administered to the subject during a3 day or longer treatment course, the agent can be administered every 2 days (i.e., every other day). In certain other embodiments, when the at least one senolytic agent is administered to the subject during a 4 day or longer course of treatment, the agent can be administered every 3 days (i.e., every third day).

Many chemotherapy and radiotherapy treatment regimens involve a limited number of cycles of drug treatment followed by drug withdrawal, or a limited time frame in which the chemotherapy or radiotherapy is administered. Such cancer treatment regimens may also be referred to as treatment regimens. The regimen can be determined by clinical trials, drug labeling, and clinical personnel in conjunction with the subject to be treated. The number of cycles of chemotherapy or radiation therapy or the total duration of chemotherapy or radiation therapy may vary depending on the patient's response to the cancer therapy. The time frame for such a treatment regimen is readily determined by one skilled in the art of oncology. In another embodiment for treating metastasis, the senolytic agent can be administered after completion of a chemotherapy or radiotherapy treatment regimen. In particular embodiments, the senolytic agent is administered one or more days within a treatment window of no more than 14 days (i.e., a senolytic agent course) after completion of the chemotherapy or radiotherapy. In other embodiments, the aging scavenger course of treatment is not longer than 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or not longer than 21 days. In other embodiments, the course of treatment is one day. In certain embodiments, the senolytic agent is administered on two or more days within a treatment window of no more than 14 days, on 3 or more days within a treatment window of no more than 14 days; (ii) administration for 4 or more days within a treatment window of no more than 14 days; (ii) administration for 5 or more days within a treatment window of no more than 14 days; administered on days 6, 7, 8, 9, 10, 11, 12, 13, or 14 within a treatment window of no more than 14 days. In certain embodiments, when the at least one senolytic agent is administered to the subject after chemotherapy or radiotherapy for a treatment window of 3 days or more, the agent may be administered every 2 days (i.e., every other day). In certain other embodiments, when at least one senolytic agent is administered to a subject for a treatment window of 4 days or longer, the agent can be administered every 3 days (i.e., every third day). In one embodiment, treatment with the senolytic agent can begin at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after completion of the cancer treatment regimen. In more specific embodiments, treatment with the senolytic agent can begin at least 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after completion of the cancer treatment regimen or thereafter. Any other course of treatment and treatment cycle of senolytic agent administration described herein can be followed to inhibit metastasis in the subject after completion of the chemotherapy or radiotherapy regimen.

Chemotherapy may be referred to as chemotherapy, chemotherapeutic agents, or chemotherapeutic drugs. Many chemotherapeutic agents are compounds known as small organic molecules. Chemotherapy is a term that is also used to describe combination chemotherapeutic drugs that are administered to treat a particular cancer. As understood by those of skill in the art, chemotherapy may also refer to a combination of two or more chemotherapeutic molecules administered synergistically, and which may also be referred to as combination chemotherapy. A variety of chemotherapy drugs are used in the field of oncology, and include, but are not limited to, alkylating agents; an antimetabolite; anthracyclines, plant alkaloids; and topoisomerase inhibitors.

The cancer that can metastasize can be a solid tumor or can be a liquid tumor (e.g., a hematologic cancer such as leukemia). Cancers of liquid tumors are classified in the art as those occurring in the blood, bone marrow and lymph nodes, and generally include leukemias (myeloid and lymphoid), lymphomas (e.g., hodgkin's lymphoma), and melanomas (including multiple myeloma). Leukemias include, for example, Acute Lymphocytic Leukemia (ALL), Acute Myelogenous Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), and hairy cell leukemia. Solid tumor cancers and cancers that occur more frequently in humans include, for example, prostate cancer, testicular cancer, breast cancer, brain cancer, pancreatic cancer, colon cancer, thyroid cancer, stomach cancer, lung cancer, ovarian cancer, kaposi's sarcoma, skin cancer (including squamous cell skin cancer), kidney cancer, head and neck cancer, throat cancer, squamous cancer formed on the moist mucosal lining of the nose, mouth, throat, etc., bladder cancer, osteosarcoma (bone cancer), cervical cancer, endometrial cancer, esophageal cancer, liver cancer, and kidney cancer. In certain particular embodiments, the disease or disorder associated with aging cells that is treated or prevented (i.e., has a reduced likelihood of occurrence or development) by the methods described herein is metastasis of melanoma cells, prostate cancer cells, testicular cancer cells, breast cancer cells, brain cancer cells, pancreatic cancer cells, colon cancer cells, thyroid cancer cells, stomach cancer cells, lung cancer cells, ovarian cancer cells, kaposi sarcoma cells, skin cancer cells, kidney cancer cells, head and neck cancer cells, throat cancer cells, squamous cancer cells, bladder cancer cells, osteosarcoma cells, cervical cancer cells, endometrial cancer cells, esophageal cancer cells, liver cancer cells, or kidney cancer cells.

The methods described herein are also useful for inhibiting, delaying or slowing the progression of metastatic cancer of any of the tumor types described in the medical field. Types of cancer (tumors) include: adrenocortical carcinoma,Childhood adrenocortical carcinoma, AIDS-related cancer, anal cancer, appendiceal cancer, basal cell carcinoma, childhood basal cell carcinoma, bladder cancer, childhood bladder cancer, bone cancer, brain tumor, childhood astrocytoma, childhood brain stem glioma, childhood central nervous system atypical teratoid/rhabdoid tumor, childhood central nervous system embryonic tumor, childhood central nervous system germ cell tumor, childhood craniopharyngioma brain tumor, childhood ependymoma brain tumor, breast cancer, childhood bronchial tumor, carcinoid tumor, childhood carcinoid tumor, gastrointestinal carcinoid tumor, carcinoma of unknown primary focus, childhood primary carcinoma of unknown origin, childhood cardiac (heart) tumor, cervical cancer, childhood chordoma, chronic myeloproliferative disease, large intestine cancer, colorectal cancer, childhood colorectal cancer, extrahepatic biliary tract cancer, hepatic biliary tract cancer, bladder cancer, childhood bladder cancer, bone cancer, brain tumor, breast, Ductal Carcinoma In Situ (DCIS), endometrial cancer, esophageal cancer, pediatric adult neuroblastoma, ocular cancer, malignant fibrous histiocytoma of bone, gallbladder cancer, gastric cancer of children, gastrointestinal stromal tumor (GIST), gastrointestinal stromal tumor of children (GIST), extracranial germ cell tumor of children, extragonadal germ cell tumor, gestational trophoblastic tumor, glioma, head and neck cancer of children, liver cell (liver) cancer, hypopharynx cancer, kidney cell kidney cancer, wilms' tumor, pediatric kidney tumor, Langerhans cell histiocytosis, laryngeal cancer of children, leukemia, Acute Lymphocytic Leukemia (ALL), Acute Myelocytic Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), chronic myelogenous leukemia (cml), Hairy cell leukemia, lip cancer, liver cancer (primary), childhood liver cancer (primary), Lobular Carcinoma In Situ (LCIS), lung cancer, non-small cell lung cancer, lymphoma, AIDS-related lymphoma, Burkitt's lymphoma, cutaneous T cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, primary central nervous system lymphoma (CNS), melanoma, childhood melanoma, intraocular (ocular) melanoma, Merkel cell carcinoma, malignant mesothelioma, childhood malignant mesothelioma, occult primary metastatic squamous cell carcinoma, cervical cancer, mid-line cancer (midline track cancer involving NUT genes), oral cancer, cervical cancer,multiple endocrine adenoma syndrome in children, mycosis fungoides, myelodysplastic syndrome, myelodysplastic tumors, myeloproliferative tumors, multiple myeloma, nasal cavity cancer, nasopharyngeal carcinoma in children, neuroblastoma, oral cavity cancer in children, oropharyngeal cancer, ovarian cancer in children, ovarian epithelial cancer, ovarian cancer of low malignancy potential tumor, pancreatic cancer in children, pancreatic neuroendocrine tumor in pancreas (islet cell tumor), papillomatosis in children, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell tumor, pleural blastoma in children, prostate cancer, rectal cancer, transitional cell carcinoma of renal pelvis, retinoblastoma, salivary gland carcinoma in children, ewing sarcoma family of tumors, Kaposi 'S sarcoma, osteosarcoma, rhabdomyosarcoma, childhood rhabdomyosarcoma, soft tissue sarcoma, uterine sarcoma, Szary' S syndrome, childhood skin cancer, non-melanoma skin cancer, small intestine cancer, squamous cell carcinoma, childhood squamous cell carcinoma, testicular cancer, childhood testicular cancer, throat cancer, thymoma and thymus cancer, childhood thymoma and thymus cancer, thyroid cancer, childhood thyroid cancer, transitional cell carcinoma of ureter, urethral cancer, endometrial uterine cancer, vaginal cancer, vulvar cancer, uterine sarcoma, neuroblastoma, uterine sarcoma, thyroid cancer, renal cancer,Macroglobulinemia.

Side effects of chemotherapy and radiotherapy. In another embodiment, the disorder or condition associated with senescent cells is a side effect of a chemotherapeutic agent or a side effect of radiation therapy. Examples of chemotherapeutic agents that induce non-cancer cell aging include anthracyclines (e.g., doxorubicin, daunorubicin); taxol (e.g., paclitaxel); gemcitabine; pomalidomide; and lenalidomide. One or more of the senolytic agents administered as described herein can be used to treat and/or prevent (i.e., reduce the likelihood of occurrence) a chemotherapeutic agent side effect or a radiation therapy side effect. Removal or destruction of senescent cells can ameliorate the acute toxicity of chemotherapy or radiotherapy, including acute toxicity comprising an energy imbalance. Acute toxic side effects include, but are not limited to, gastrointestinal toxicity (e.g., nausea)Heart, vomiting, constipation, anorexia, diarrhea), peripheral neuropathy, fatigue, restlessness, decreased physical activity, hematologic toxicity (e.g., anemia), hepatotoxicity, alopecia (alopecia), pain, infection, mucositis, fluid retention, skin toxicity (e.g., rash, dermatitis, pigmentation, urticaria, photosensitivity, nail changes), mouth (e.g., oral mucositis), gum or throat problems, or any toxic side effects resulting from chemotherapy or radiation therapy. For example, toxic side effects resulting from radiation or chemotherapy (see, e.g., the national cancer institute website) can be mitigated by the methods described herein. Thus, in certain embodiments, methods are provided for reducing (reducing, inhibiting, or preventing the occurrence of (i.e., reducing the likelihood of occurrence)) acute toxicity, or reducing the severity of toxic side effects (i.e., deleterious side effects) of chemotherapy or radiotherapy, or both, in a subject receiving such therapy, wherein the method comprises administering to the subject an agent that selectively kills, eliminates, or destroys, or promotes selective destruction of, aging cells. Administration of senolytic agents to treat or reduce the likelihood of occurrence, or reduce the severity of side effects of chemotherapy or radiotherapy can be accomplished by the same course of treatment as described above for treatment/prevention of metastasis. Senolytic agents are administered during a non-chemotherapeutic or non-radiotherapeutic interval or after completion of a chemotherapeutic or radiotherapeutic treatment regimen, as described for treating or preventing (i.e., reducing the likelihood of occurrence of) metastasis.

In more specific embodiments, the acute toxicity is an acute toxicity comprising an energy imbalance, and may include one or more of weight loss, endocrine changes (e.g., hormonal imbalance, hormonal signaling changes), and changes in body composition. In certain embodiments, acute toxicity comprising an energy imbalance is associated with a decrease or reduction in physical activity of the subject, as indicated by a decrease or reduction in energy expenditure as compared to that which would be observed in a subject not receiving the medical therapy. By way of non-limiting example, such acute toxic effects, including energy imbalances, include decreased physical mobility. In other particular embodiments, the energy imbalance includes fatigue or restlessness.

In one embodiment, the side effect of chemotherapy to be treated or prevented (i.e., reduce the likelihood of occurrence) by the senolytic agent is cardiotoxicity. Anthracycline-treated subjects with cancer (e.g., doxorubicin, daunorubicin) can be treated with one or more senolytic agents that reduce, alleviate, or reduce the cardiotoxicity of the anthracycline. As is well known in the medical arts, the maximum lifetime dose that a subject can receive is limited due to the cardiotoxicity associated with anthracycline drugs, even if the cancer is responsive to the drug. Administration of one or more senolytic agents can reduce cardiotoxicity such that additional amounts of the anthracycline can be administered to the subject, resulting in an improvement in prognosis associated with the cancer disease. In one embodiment, the cardiotoxicity results from administration of an anthracycline, such as doxycycline. Doxorubicin is an anthracycline topoisomerase approved for use in treating patients with ovarian cancer after failure of platinum-based therapy; for treating patients with kaposi's sarcoma after failure of primary systemic chemotherapy or resistance to that therapy; or in combination with bortezomib for the treatment of patients who have not previously received bortezomib or who have received at least one prior therapy. Doxorubicin can cause myocardial damage if the total lifetime dose of the patient exceeds 550mg/m²This myocardial damage can lead to congestive heart failure. If the patient also receives mediastinal irradiation or another cardiotoxic drug, cardiotoxicity may occur at even lower doses. See pharmaceutical product inserts (e.g., DOXIL, ADRIAMYCIN).

In other embodiments, the senolytic agents described herein can be used in a method as provided herein to ameliorate chronic or long-term side effects. Chronic side effects are often caused by administration or multiple exposures to chemotherapy or radiation therapy over a prolonged period of time. Some toxic effects occur relatively long after treatment (also known as late toxic effects) and are due to damage to the organ or system from the therapy. Organ dysfunction (e.g., neurological, pulmonary, cardiovascular, and endocrine dysfunction) has been observed in patients treated for cancer in childhood (see, e.g., Hudson et al, JAMA 309:2371-81 (2013)). Without wishing to be bound by any particular theory, by destroying senescent cells, specific normal cells that have been induced to senesce by chemotherapy or radiotherapy, the likelihood of the onset of chronic side effects may be reduced, or the severity of chronic side effects may be reduced or attenuated, or the time to onset of chronic side effects may be delayed. By way of non-limiting example, chronic and/or late toxic side effects that occur in subjects receiving chemotherapy or radiation therapy include cardiomyopathy, congestive heart disease, inflammation, premenopausal phase, osteoporosis, infertility, impaired cognitive function, peripheral neuropathy, secondary cancer, cataracts and other vision problems, hearing loss, chronic fatigue, decreased lung capacity, and pulmonary disease.

In addition, by killing or removing senescent cells in a subject with cancer via administration of a senolytic agent, the sensitivity to chemotherapy or radiation therapy is clinically or statistically significantly increased compared to the situation without administration of a senolytic agent. In other words, when the senolytic agent is administered to a subject treated with the respective chemotherapy or radiotherapy, development of chemotherapy or radiotherapy resistance can be inhibited.

Age-related diseases and disorders.Senolytic agents can also be used to treat or prevent (i.e., reduce the likelihood of occurrence of) an age-related disease or disorder that occurs as part of the natural aging process or when a subject is exposed to a senescence-inducing agent or factor (e.g., radiation, chemotherapy, tobacco smoking, high fat/high sugar diet, other environmental factors). The nature of the age-related condition or disease or age-sensitivity may be related to aging-induced stimuli. The efficacy of the therapeutic methods described herein can be evidenced by a reduction in the number of symptoms, a reduction in the severity of one or more symptoms, or a delay in the progression of an age-related disorder or an age-sensitive trait associated with aging-induced stimuli. In other particular embodiments, preventing an age-related disorder or an age-sensitive trait associated with senescence-inducing stimuli refers to preventing (i.e., reducing the likelihood of occurrence) or delaying an age-related disorder associated with senescence-inducing stimuliOr the onset of an age-sensitive trait, or the recurrence of one or more age-related conditions or age-sensitive traits associated with aging-induced stimuli. Age-related diseases or conditions include, for example, renal dysfunction, kyphosis, herniated intervertebral disc, weakness, hair loss, hearing impairment, vision impairment (blindness or impaired vision), muscle fatigue, skin conditions, skin nevi, diabetes, metabolic syndrome, and sarcopenia. Visual impairment refers to the lack of vision in a subject who has had prior vision. Various scales have been developed for describing vision and the degree of vision impairment based on visual acuity. Age-related diseases and conditions also include skin diseases, such as, but not limited to, treatment of one or more of the following conditions: wrinkles, including superficial fine lines; hyperpigmentation; scars; keloid scars; dermatitis; psoriasis; eczema (including seborrheic eczema); rosacea; vitiligo; ichthyosis vulgaris; dermatomyositis; and actinic keratosis.

Frailty has been defined as a clinically recognizable state of increased vulnerability caused by age-related reductions in reserves and functions across multiple physiological systems that reduce the ability of a subject to cope with daily or acute stress factors. Weakness may be characterized by impaired energy characteristics such as low grip strength, low energy, slow walking speed, low physical activity and/or unexpected weight loss. Studies have shown that patients can be diagnosed with frailty when three of the five above-described features are observed (see, e.g., Fried et al, j. gerntol. a biol. sci. med, sci.200156(3): M146-M156; Xue, clin. geriatr. med. 2011; 27(1): 1-15). In certain embodiments, aging and diseases and disorders associated with aging can be treated or prevented (i.e., the likelihood of occurrence is reduced) by administering a senolytic agent. The senolytic agent can inhibit senescence or inhibit accumulation of adult stem cells, kill adult stem cells that have become senescent, or promote their removal. See, e.g., Park et al, j.clin.invest.113: 175-79(2004) and Sousa-Victor, Nature 506:316-21(2014), which describe the importance of preventing senescence of stem cells in maintaining the regenerative capacity of a tissue.

The effectiveness of a senolytic agent for treating a senescence-associated disease or disorder described herein can be readily determined by one of skill in the medical and clinical arts. One or any combination of diagnostic methods known to those skilled in the art (including physical examination, patient self-assessment, assessment and monitoring of clinical symptoms, performance of analytical tests and procedures including, for example, clinical laboratory tests, physical performance tests, and exploratory surgery) suitable for use in the particular disease or condition may be used to monitor the health status of the subject and the effectiveness of the senolytic agent. The effect of the treatment methods described herein can be analyzed using techniques known in the art, such as comparing the symptoms of a patient who has received a pharmaceutical composition comprising a senolytic agent, who is suffering from or at risk of a particular disease or disorder, to the symptoms of a patient who is not being treated with a senolytic agent or who is receiving placebo treatment.

As understood by those of skill in the Medical arts, the terms "treatment" and "treating" refer to the Medical control of a disease, disorder, or condition in a subject (i.e., patient) (see, e.g., Stedman's Medical Dictionary). In general, the appropriate dosage and treatment regimen provides the senolytic agent in an amount sufficient to provide a therapeutic and/or prophylactic benefit. Therapeutic benefits of a subject administered a senolytic agent as described herein include, for example, improved clinical outcome, wherein the objective is to prevent or slow down or delay (lessen) undesired physiological changes associated with the disease, or prevent or slow down or delay (lessen) the expansion or severity of such disease. As discussed herein, the effectiveness of one or more senolytic agents can include beneficial or desired clinical results including, but not limited to, reduction, alleviation, or alleviation of symptoms caused by or associated with the disease to be treated; a reduction in the occurrence of symptoms; the quality of life is improved; longer disease-free states (i.e., a reduced likelihood or propensity that a subject will have symptoms based on a diagnosis of the disease); a reduced degree of disease; stable disease state (i.e., not deteriorating); delay or slowing of disease progression; alleviation or remission of the disease state; and mitigation (whether partial or total), whether detectable or undetectable; and/or overall lifetime. The effectiveness of a senolytic agent as described herein can also refer to an extended survival time as compared to the survival time expected if the subject did not receive a senolytic agent that selectively kills senescent cells.

Administration of the senolytic agents described herein can extend survival compared to survival expected if the subject is not receiving treatment. Subjects in need of treatment include those already with the disease or disorder, as well as subjects susceptible to or at risk of developing the disease or disorder, as well as those who will be prophylactically treated for the disease, condition, or disorder. The subject may have a genetic predisposition to develop a disease or condition that would benefit from the clearance of senescent cells, or may be of an age at which receiving an aging scavenger would provide a clinical benefit to delay disease progression or reduce the severity of a disease, including age-related diseases or conditions.

In another embodiment, a method for treating a senescence-associated disease or disorder is provided, which further comprises identifying a subject that would benefit from treatment with a senolytic agent described herein (i.e., phenotypic analysis; personalized treatment). The method comprises first detecting the level of senescent cells in a subject, such as in particular in an organ or tissue of the subject. A biological sample can be obtained from a subject, e.g., a blood sample, serum or plasma sample, a biopsy specimen, a bodily fluid (e.g., lung lavage fluid, ascites fluid, mucosal washes, synovial fluid, vitreous humor, spinal fluid), bone marrow, lymph nodes, tissue explants, organ cultures, or any other tissue or cell preparation from a subject. The level of senescent cells can be determined according to any of the in vitro assays or techniques described herein. For example, the biological sample can be obtained by morphology (e.g., as observed by microscopy); senescence-associated markers such as the production of senescence-associated β -galactosidase (SA- β -gal), p16INK4a, p21, PAI-1, or any one or more of the SASP factors (e.g., IL-6, MMP 3). Senescent cells and non-senescent cells of a biological sample can also be used in an in vitro cellular assay, wherein cells are exposed to any of the senolytic agents described herein to determine the ability of the senolytic agent to kill senescent cells of a subject without undesirable toxicity to non-senescent cells. As a positive control in these assays, the assay may comprise any of the senolytic agents described herein (e.g., Nutlin-3a, RG-7112, ABT-263, ABT-737, WEHI-539, A-1155463, MK-2206). The subject may then be treated with an appropriate senolytic agent, which may be an MDM2 inhibitor; an inhibitor of one or more Bcl-2 anti-apoptotic protein family members, wherein the inhibitor inhibits at least Bcl-xL (e.g., a Bcl-xL selective inhibitor, a Bcl-2/Bcl-xL/Bcl-w inhibitor, a Bcl-2/Bcl-xL or a Bcl-xL/Bcl-w inhibitor); or specific inhibitors of Akt. In addition, these methods can also be used to monitor the level of senescent cells in a subject before, during, and after treatment of the subject with a senolytic agent. In certain embodiments, the presence of senescent cells can be detected (e.g., by determining the level of expression of senescent cell markers, such as mRNA), and the course of treatment and/or the absence of treatment intervals can be adjusted accordingly.

A subject, patient, or individual in need of treatment with a senolytic agent as described herein can be a human, or can be a non-human primate or other animal (i.e., veterinary) that has developed symptoms of, or is at risk of developing, a senescent cell-related disease or disorder. Non-human animals that can be treated include mammals, e.g., non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pigs, piglets), horses, dogs, cats, cows, elephants, bears, and other domestic, farm, and zoo animals.

Methods for characterizing and identifying senolytic agents

Characterizing senolytic agents can be determined using one or more cell-based assays and one or more animal models described herein or well known in the art. A senolytic agent is an agent that selectively kills or destroys senescent cells in a statistically, clinically, or biologically significant manner. Senolytic agents can selectively kill one or more types of senescent cells (e.g., senescent preadipocytes, senescent endothelial cells, senescent fibroblasts, senescent neurons, senescent epithelial cells, senescent mesenchymal cells, senescent smooth muscle cells, senescent macrophages, or senescent chondrocytes). In certain particular embodiments, the senolytic agent is capable of at least selectively killing senescent fibroblasts.

Characterizing an agent, such as a senolytic agent, can be accomplished using one or more cell-based assays and one or more animal models described herein or in the art. One skilled in the art will readily appreciate that characterizing an agent, such as a senolytic agent, and determining the level of killing by the agent, can be achieved by comparing the activity of the test agent to a suitable negative control (e.g., vehicle or diluent only, and/or a composition or compound known in the art that does not kill senescent cells) and a suitable positive control. In vitro cell-based assays for characterizing senolytic agents also include controls for determining the effect of the agent on non-senescent cells (e.g., resting cells or proliferating cells). Senolytic agents reduce (i.e., decrease) the percent survival of a plurality of senescent cells (i.e., decrease the amount of viable senescent cells in an animal or cell-based assay in some manner) compared to one or more negative controls. Specific in vitro assay conditions include temperature, buffers (including salts, cations, media), and other components that will maintain the integrity of the test agents and reagents used in the assay and are familiar to and/or readily determinable by one of skill in the art.

The source of senescent cells for the assay can be a primary cell culture or culture adapted cell line, including, but not limited to, genetically engineered cell lines that can contain chromosomally integrated or episomally recombined nucleic acid sequences, immortalized or immortalized cell lines, somatic hybrid cell lines, differentiated or differentiable cell lines, transformed cell lines, and the like. In particular embodiments, the senescent cells are isolated from a biological sample, wherein the biological sample is obtained from a host or subject having a disease or disorder associated with senescent cells. In other embodiments, non-senescent cells that can be obtained from a subject or can be a culture-adapted cell line can be used and can induce senescence by methods described herein and in the art, such as by exposure to radiation or a chemotherapeutic agent (e.g., doxorubicin). The biological sample can be a blood sample, biopsy specimen, bodily fluid (e.g., lung lavage fluid, ascites fluid, mucosal washes, synovial fluid), bone marrow, lymph node, tissue explant, organ culture, or any other tissue or cell preparation from a subject. The sample may be a tissue or cell preparation in which the morphological integrity or physical state has been disrupted by, for example, dissection, dissociation, lysis, fractionation, homogenization, biochemical or chemical extraction, comminution, freeze drying, sonication, or any other means for processing a sample derived from a subject or biological source. The subject may be a human or non-human animal.

Transgenic animal models as described herein and in the art can be used to determine the killing or removal of senescent cells (see, e.g., Baker et al, supra; Nature,479:232-36 (2011); International patent application publication No. WO/2012/177927; International patent application publication No. WO 2013/090645). Exemplary transgenic animal models contain transgenes comprising senescence-permitting cells (e.g., p16)^ink4aPositive senescent cells) as a positive control. The presence and level of senescent cells in a transgenic animal can be determined by measuring the level of a detectable marker expressed in senescent cells of the animal. The transgenic nucleotide sequence comprises a detectable label, e.g., one or more red fluorescent proteins; green fluorescent protein; and one or more luciferases to detect clearance of senescent cells.

Animal models described herein or in the art include art-accepted models, such as an atherosclerosis model, an osteoarthritis model, a COPD model, and an IPF model, for determining the effectiveness of a senolytic agent for treating or preventing (i.e., reducing the likelihood of occurrence of) a particular senescence-associated disease or disorder. As described herein, pulmonary murine models, such as bleomycin pulmonary fibrosis models and chronic smoking models, may be applicable to diseases such as COPD, and may be routinely practiced by those of skill in the art. Animal models for determining the efficacy of senolytic agents for the treatment and/or prevention (i.e., reducing the likelihood of occurrence) of chemotherapy and radiotherapy side effect models, or for the treatment or prevention (i.e., reducing the likelihood of occurrence) of metastasis, are described in international patent application publication nos. WO2013/090645 and WO 2014/205244, which are incorporated herein by reference in their entirety. Animal models for determining the effectiveness of agents for treating ocular diseases, particularly age-related macular degeneration, are also routinely used in the art (see, e.g., pennisi et al, mol. aspects med. 33: 487-.

By way of non-limiting example and as described herein, animal models of osteoarthritis have been developed. Osteoarthritis can be induced in an animal by, for example, inducing damage to a joint, for example, in the knee by surgical partial or complete severing of the anterior cruciate ligament. The osteoarthritic animal model can be used to evaluate the effectiveness of senolytic agents for treating or preventing (i.e., reducing the likelihood of occurrence) osteoarthritides and resulting in decreased proteoglycan erosion, and for inducing (i.e., stimulating, enhancing) collagen (e.g., type 2 collagen) production, and for reducing pain in animals having ACL surgery. Immunohistology can be performed to examine the integrity and composition of tissues and cells in the joint. Immunohistological and/or molecular biological techniques, such as assays for determining the level of inflammatory molecules (e.g., IL-6) and assays for determining the level of aging markers as mentioned above, can also be performed using the methods and techniques described herein and which can be routinely practiced by those of skill in the art.

By way of another non-limiting example and as described herein, animal models of atherosclerosis have been developed. Atherosclerosis can be induced in an animal by, for example, feeding the animal a high fat diet, or by using a transgenic animal that is highly susceptible to developing atherosclerosis. Animal models can be used to determine the effectiveness of senolytic agents for reducing the amount of plaque or for inhibiting plaque formation in atherosclerotic arteries, for reducing the lipid content of atherosclerotic plaque (i.e., reducing, decreasing the amount of lipid in plaque), and for increasing the fibrous cap thickness of plaque or causing an increase in plaque thereof. Sudan staining can be used to detect lipid levels in atherosclerotic blood vessels. Immunohistological and immunochemical and molecular biological assays (e.g., for determining levels of inflammatory molecules (e.g., IL-6) and for determining levels of aging markers as mentioned above) can all be performed according to the methods described herein and routinely practiced in the art.

In yet another example, and as described herein, a mouse model for determining the effectiveness of an agent for treating IPF, wherein the animal is treated with bleomycin, has been described (see, e.g., Peng et al, PLoS One 2013; 8(4): e59348.doi: 10.1371/journal. po. 0059348.epub 2013Apr 2; mourvatis et al, curr. opin. palm. med.17:355-61 (2011)). In animal models of pulmonary disease (e.g., bleomycin animal model, smoke exposure animal model, etc.), respiratory measurements can be taken to determine elasticity (elastance), compliance, static compliance, and peripheral capillary blood oxygen saturation (SpO)₂). Immunohistological and immunochemical and molecular biological assays (e.g., for determining levels of inflammatory molecules (e.g., IL-6) and for determining levels of aging markers as mentioned above) can all be performed according to the methods described herein and routinely practiced in the art.

Determination of the effectiveness of senolytic agents for selectively killing senescent cells as described herein in animal models can be performed using one or more statistical analyses familiar to the skilled artisan. By way of example, statistical analyses such as two-way analysis of variance (ANOVA) can be used to determine the statistical significance of the differences between the groups of animals treated with the agent and those not treated with the agent (i.e., the negative control group, which may contain only vehicle and/or non-senolytic agent). Statistical software packages such as SPSS, MINITAB, SAS, Statistika, Graphpad, GLIM, Genstat, and BMDP are readily available and routinely used by those skilled in the art of animal models.

One skilled in the art will readily appreciate that characterizing a senolytic agent and determining the level of killing by the senolytic agent can be achieved by comparing the activity of the test agent to a suitable negative control (e.g., vehicle only, and/or a composition, agent, or compound known in the art that does not kill senescent cells) and a suitable positive control. In vitro cell-based assays for characterizing the agent also include controls for determining the effect of the agent on non-senescent cells (e.g., resting cells or proliferating cells). Useful senolytic agents reduce (i.e., decrease) the percent survival of senescent cells (i.e., decrease the amount of viable senescent cells in an animal or cell-based assay in some manner) compared to one or more negative controls. Thus, a senolytic agent will selectively kill senescent cells compared to the killing of non-senescent cells (which may be referred to herein as selectively killing senescent cells rather than non-senescent cells). In certain embodiments (in an in vitro assay or in an in vivo assay (in a human or non-human animal)), the at least one senolytic agent kills at least 20% of senescent cells and kills no more than 5% of non-senescent cells. In other particular embodiments (in an in vitro assay or in an in vivo assay (in a human or non-human animal)), the at least one senolytic agent kills at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% of the senescent cells and kills no more than about 5% or 10% of the non-senescent cells. In other particular embodiments (in an in vitro assay or in an in vivo assay (in a human or non-human animal)), the at least one senolytic agent kills at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% of senescent cells and kills no more than about 5%, 10%, or 15% of non-senescent cells. In other particular embodiments (in an in vitro assay or in an in vivo assay (in a human or non-human animal)), the at least one senolytic agent kills at least about 40%, 45%, 50%, 55%, 60%, or 65% of senescent cells and kills no more than about 5%, 10%, 15%, 20%, or 25% of non-senescent cells. In other particular embodiments (in an in vitro assay or in an in vivo assay (in a human or non-human animal)), the at least one senolytic agent kills at least about 50%, 55%, 60%, or 65% of senescent cells and kills no more than about 5%, 10%, 15%, 20%, 25%, or 30% of non-senescent cells. In other words, the senolytic agent has a selectivity (e.g., at least 5x, 10x, 20x, 25x, 30x, 40x, 50x, 60x, 75x, 80x, 90x, or 100x) for killing senescent cells of at least 5-25, 10-50, or 10-100 fold (5x-25x, 10x-50x, or 10 x-100 x) over non-senescent cells. For particular embodiments of the methods described herein for treating a disease or disorder associated with aging, the percentage of aged cells killed can refer to the percentage of aged cells killed in a tissue or organ containing aged cells that cause the disease or disorder to develop, progress, and/or aggravate. By way of non-limiting example, tissues of the brain, tissues and parts of the eye, lung tissue, heart tissue, blood vessels, joints, skin and muscle may comprise senescent cells that can be reduced by the senolytic agents described herein by a percentage as described above and thereby provide a therapeutic effect. Moreover, selective removal of at least 20% or at least 25% of senescent cells from an infected organ or tissue can have a clinically significant therapeutic effect. For particular embodiments of the methods described herein (e.g., for treating a cardiovascular disease or disorder associated with arteriosclerosis, such as atherosclerosis), by administering a senolytic agent (i.e., with reference to the in vivo methods described above), the percentage of senescent cells killed can refer to the percentage of senescent cells killed in an infected artery containing plaque relative to non-senescent cells killed in an arterial plaque. In certain particular embodiments, in the methods for treating a cardiovascular disease as described herein, such as atherosclerosis, the at least one senolytic agent kills at least 20% of the senescent cells and kills no more than 5% of the non-senescent cells in the artery. In other particular embodiments, the senolytic agent selectively kills at least 25% of the senescent cells in the arteriosclerotic arteries. In another embodiment, for the methods described herein for treating osteoarthritis by administration of a senolytic agent, the percentage of senescent cells killed can refer to the percentage of senescent cells killed in an osteoarthritic joint relative to non-senescent cells killed in an osteoarthritic joint. In certain particular embodiments, in the method for treating osteoarthritis as described herein, the at least one senolytic agent kills at least 20% of the senescent cells and kills no more than 5% of the non-senescent cells in the osteoarthritic joint. In other particular embodiments, the senolytic agent selectively kills at least 25% of the senescent cells in the osteoarthritic joint. In yet another embodiment, for the methods described herein for treating a senescence-associated lung disease or disorder (e.g., COPD, IPF) by administering at least one senolytic agent, the percentage of senescent cells killed can refer to the percentage of senescent cells killed in infected lung tissue relative to non-senescent cells killed in infected lung tissue of the lung. In certain particular embodiments, in the methods for treating senescence-associated lung diseases and disorders as described herein, the senolytic agent kills at least 20% of senescent cells and kills no more than 5% of non-senescent cells in infected lung tissue. In other particular embodiments, the senolytic agent selectively kills at least 25% of the senescent cells in the infected lung tissue.

In certain embodiments, methods are provided for identifying (i.e., screening) senolytic agents that are useful for treating or preventing (i.e., reducing the likelihood of occurrence of) a senescence-associated disease or disorder. In one embodiment, a method for identifying senolytic agents for treating such diseases and disorders comprises inducing cellular senescence to provide defined senescent cells. Methods for inducing cellular senescence are described herein and in the art, and include, for example, exposure to radiation (e.g., 10Gy is typically sufficient) or chemotherapeutic agents (e.g., doxorubicin or other anthracyclines). After exposure to the agent, the cells are subjected to appropriate conditions (e.g., medium, temperature, CO appropriate for a given cell type or cell line)₂/O₂Horizontal) for a suitable time to allow senescence to develop. As discussed herein, senescence of cells can be determined by any number of characteristics, such as morphological changes (e.g., as observed by microscopy); such as the production of senescence-associated beta-galactosidase (SA-beta-gal), p16INK4a, p21, or any one or more of the SASP factors (e.g., IL-6, MMP 3). Then the aged cells areThe sample is contacted with (i.e., mixed with, combined with, or in some embodiments allowed to interact with) the candidate agent. One skilled in the art will appreciate that the assay will include appropriate negative and positive controls, either historical or performed simultaneously. For example, a sample of control non-senescent cells that have been cultured similarly to senescent cells but not exposed to a senescence-inducing agent is contacted with the candidate agent. The level of survival of senescent cells is determined and compared to the level of survival of non-senescent cells. Senolytic agents are identified when the level of survival of senescent cells is lower than the level of survival of non-senescent cells.

In certain embodiments, the above-described method of identifying a senolytic agent may further comprise a step for identifying whether the senolytic agent is useful for treating osteoarthritis. The method may further comprise contacting the identified senolytic agent with a cell capable of producing collagen; and determining the level of collagen produced by the cell. In a particular embodiment, the cells are chondrocytes and the collagen is type 2 collagen. The method may further comprise administering the candidate senolytic agent to a non-human animal having arthritis loss in a joint and determining one or more of: (a) the level of senescent cells in the joint; (b) physical function of the animal; (c) the level of one or more markers of inflammation; (d) histology of the joint; and (e) the level of collagen type 2 produced, thereby determining the therapeutic efficacy of the senolytic agent, wherein one or more of the following is observed in the treated animal compared to an animal not treated with the senolytic agent: (i) treating a decrease in the level of senescent cells in the joints of the animal; (ii) treating an improvement in bodily function of the animal; (iii) treating a reduction in the level of one or more markers of inflammation in an animal; (iv) treating an increase in the histological normality of the joints of the animal; and (v) an increase in the level of type 2 collagen produced in the treated animal. As described herein and in the art, the physical function of an animal can be determined by techniques that determine the sensitivity of the legs to induced or natural osteoarthritic conditions by, for example, the animal's tolerance to weight bearing on an infected limb, or the animal's ability to move away from an intractable stimulus such as heat or cold. Determination of the efficacy of an agent to kill senescent cells as described herein in an animal model can be made using one or more statistical analyses familiar to the skilled artisan. Statistical analysis as described herein and as routinely practiced in the art can be used to analyze the data.

In another particular embodiment, the above method of identifying a senolytic agent may further comprise a step for identifying whether the senolytic agent is useful for treating a cardiovascular disease caused by or associated with arteriosclerosis. Thus, the method may further comprise administering the senolytic agent candidate agent in a non-human animal of an animal model to determine the effectiveness of the agent for reducing the amount of plaque, for arresting plaque formation in an atherosclerotic artery, for reducing the lipid content of an atherosclerotic plaque (i.e., reducing, decreasing the amount of lipid in a plaque), and/or for increasing the fibrous cap thickness of a plaque or causing an increase in its plaque. Sudan staining can be used to detect lipid levels in atherosclerotic blood vessels. Immunohistology, assays for determining levels of inflammatory molecules (e.g., IL-6), and/or assays for determining levels of aging markers as mentioned above can all be performed according to the methods described herein and routinely practiced in the art. In particular embodiments, the methods described herein for identifying senolytic agents can further comprise administering a candidate senolytic agent to a non-human animal having an atherosclerotic plaque and determining one or more of: (a) the level of senescent cells in the arteries; (b) physical function of the animal; (c) the level of one or more markers of inflammation; (d) histology of infected vessels (arteries); and determining therefrom the therapeutic efficacy of the senolytic agent, wherein one or more of the following is observed in the treated animal compared to an animal not treated with the senolytic agent: (i) treating a decrease in the level of senescent cells in an artery of an animal; (ii) treatment of improvement in physical function in an animal; (iii) treating a reduction in the level of one or more markers of inflammation in an animal; (iv) the histologic normality in the arteries of the treated animals was increased. As described herein and in the art, the physical function of an animal can be determined by measuring physical activity. Statistical analysis as described herein and as routinely practiced in the art can be used to analyze the data.

In one embodiment, the methods described herein for identifying senolytic agents can comprise administering a candidate senolytic agent to a non-human animal lung disease model, such as a bleomycin model or a smoke exposure animal model, and determining one or more of: (a) the level of senescent cells in the lung; (b) pulmonary function of the animal; (c) the level of one or more markers of inflammation; (d) histology of lung tissue, thereby determining the therapeutic efficacy of the senolytic agent, wherein one or more of the following is observed in the treated animal compared to an animal not treated with the senolytic agent: (i) treating the reduction in the level of senescent cells in the lung and lung tissue of the animal; (ii) treating an improvement in lung function in an animal; (iii) treating a reduction in the level of one or more markers of inflammation in an animal; and (iv) treating the increase in histologically normal status in lung tissue of the animal. Respiratory measurements may be taken to determine elasticity (elastance), compliance, static compliance, and peripheral capillary blood oxygen saturation (SpO)₂). Pulmonary function can be assessed by determining any of a variety of measures, such as, for example, supplementary expiratory volume (ERV), Forced Vital Capacity (FVC), Forced Expiratory Volume (FEV) (e.g., FEV in one second, FEV1), FEV1/FEV ratio, forced expiratory flow 25% to 75%, and maximum ventilation volume (MVV maximum expiratory flow (PEF)), Slow Vital Capacity (SVC). Total lung volume includes total lung volume (TLC), Vital Capacity (VC), residual capacity (RV), and Functional Residual Capacity (FRC). Carbon monoxide diffusion capacity (DLCO) can be used to measure gas exchange across the alveolar-capillary membrane. Peripheral capillary blood oxygen saturation (SpO) can also be measured₂). Statistical analysis as described herein and as routinely practiced in the art can be used to analyze the data.

In vitro and in vivo assays (e.g., animal models) described herein for identifying and characterizing senolytic agents can include any of the senolytic agents described herein (e.g., Nutlin-3a, RG-7112, ABT-263, ABT-737, WEHI-539, A-1155463, MK-2206) as positive controls. Specific in vitro assay conditions include temperature, buffers (including salts, cations, media), and other components (e.g., cells) that maintain the integrity of the test agents and reagents used in the assay and are well known and/or readily ascertainable by those skilled in the art. The assays and techniques described herein can also be used in toxicology analysis methods, quality control assays, and the like, routinely performed during drug development and used for quality assurance. The animal models for these methods and purposes may include non-human primate models, dog models, rodent models, or other animal models suitable for determining the safety and efficacy of senolytic agents.

Pharmaceutical composition

Also provided herein are pharmaceutical compositions comprising a senolytic agent (e.g., a MDM2 inhibitor; an inhibitor of one or more Bcl-2 anti-apoptotic protein family members, wherein the inhibitor at least inhibits Bcl-xL (e.g., a Bcl-xL selective inhibitor, a Bcl-2/Bcl-xL/Bcl-w inhibitor, a Bcl-2/Bcl-xL or a Bcl-xL/Bcl-w inhibitor), or an Akt-specific inhibitor) as described herein and at least one pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient can also be referred to as a pharmaceutically suitable excipient or carrier (i.e., a non-toxic substance that does not interfere with the activity of the active ingredient). The pharmaceutical compositions may be sterile aqueous or non-aqueous solutions, suspensions or emulsions (e.g., microemulsions). The excipients described herein are examples and are not limiting at all. An effective or therapeutically effective amount refers to an amount of one or more senolytic agents administered to a subject that is effective to produce a desired therapeutic effect as a single dose or as part of a series of doses.

When two or more senolytic agents are administered to a subject for treating a disease or disorder described herein, each senolytic agent can be formulated as a separate pharmaceutical composition. Pharmaceutical formulations may be prepared comprising each of the separate pharmaceutical compositions (which may be referred to for convenience as, for example, the first and second pharmaceutical compositions comprising each of the first and second senolytic agents, respectively). Each pharmaceutical composition in the formulation may be administered at the same time (i.e., simultaneously) by the same route of administration, or may be administered at different times by the same or different routes of administration. Alternatively, two or more senolytic agents may be formulated together in a single pharmaceutical composition.

In other embodiments, a combination of at least one senolytic agent and at least one inhibitor of the mTOR, nfkb, or PI3-k pathway may be administered to a subject in need thereof. When at least one senolytic agent is used in the methods described herein together with at least one inhibitor of the mTOR, nfkb, or PI3-k pathway to selectively kill senescent cells, each agent may be formulated as the same pharmaceutical composition or as separate pharmaceutical compositions. Pharmaceutical formulations may be prepared comprising each of the individual pharmaceutical compositions, wherein each of the individual pharmaceutical compositions may be referred to for convenience as, for example, a first pharmaceutical composition and a second pharmaceutical composition comprising an senolytic agent and each of at least one inhibitor of the mTOR, nfkb, or PI3-k pathway, respectively. Each of the pharmaceutical compositions in the formulation may be administered at the same time by the same route of administration, or may be administered at different times by the same or different routes of administration.

In particular embodiments, a single senolytic agent is administered to a subject, and it is a single (i.e., the only) active senolytic agent (i.e., a single therapy) for treating a condition or disease. When the senolytic agent is a single senolytic agent, the use of a drug for other purposes, such as a palliative drug or a drug used for comfort, is not necessarily excluded; or drugs for treating specific diseases or conditions but not senolytic agents, such as cholesterol-lowering drugs or ocular wetting agents, and other such drugs familiar to those skilled in the medical arts. By way of non-limiting example, examples of other agents and drugs that may be administered to a subject with a pulmonary disease (e.g., COPD) include bronchodilators (e.g., anticholinergic drugs; beta-2 agonists); an analgesic; medicaments and drugs that may be administered to a subject suffering from osteoarthritis include hyaluronic acid, analgesic drugs (including topical drugs), and steroids. Other agents and drugs that may be administered to subjects with cardiovascular disease include statins, beta blockers, nitroglycerin, aspirin.

The effectiveness of a treatment in a subject can generally be monitored using assays and methods suitable for the condition to be treated, which assays will be familiar to those of ordinary skill in the art and are described herein. The pharmacokinetics of the senolytic agent (or one or more metabolites thereof) administered to a subject can be monitored by determining the level of the senolytic agent in a biological fluid, such as blood, blood components (e.g., serum), and/or urine, and/or other biological sample or biological tissue from the subject. The practice in the art for detecting such agents and any of the methods described herein can be used to measure the level of senolytic agent during a course of treatment.

The dosage of the senolytic agent described herein for treating a senescent cell-associated disease or disorder can depend on the condition of the subject, i.e., the stage of the disease, the severity of the symptoms caused by the disease, the general health, as well as age, sex, and weight, as well as other factors apparent to those of skill in the medical arts. The pharmaceutical composition may be administered in a manner appropriate to the disease to be treated as determined by one of skill in the medical arts. In addition to the factors described above with respect to the use of senolytic agents for the treatment of a senolytic-related disease or disorder, the appropriate duration and frequency of senolytic agent administration may be determined or adjusted by factors such as the condition of the patient, the type and severity of the patient's disease, the particular form of the active ingredient, and the method of administration. Experimental models and/or clinical trials can generally be used to determine the optimal dosage of an agent. The optimal dosage may depend on the body mass, weight or blood volume of the subject. It is generally preferred to use a minimum amount sufficient to provide effective treatment. The design and implementation of preclinical and clinical studies (including when administered for prophylactic benefit) of the senolytic agents described herein is well within the skill of one of ordinary skill in the relevant art. When two or more senolytic agents are administered to treat a senescence-associated disease or disorder, the optimal dose of each senolytic agent can be different, e.g., lower than the dose of each agent when administered alone as a single agent therapy. In certain particular embodiments, the two senolytic agents act in combination synergistically or additively, such that each agent can be used in lower amounts than if administered alone. For example, the amount of senolytic agent that can be administered daily can be between about 0.01mg/kg and 100mg/kg (e.g., between about 0.1 and 1 mg/kg), between about 1 and 10mg/kg, between about 10-50 mg/kg, between about 50-100mg/kg, by weight. In other embodiments, the amount of senolytic agent that can be administered daily is between about 0.01mg/kg and 1000mg/kg, between about 100 and 500mg/kg, or between about 500 and 1000mg/kg, by weight. In particular embodiments, the total amount of MDM2 inhibitor (e.g., Nutlin-3a) per treatment cycle, the total amount of senilisation scavenger administered per course of treatment, does not exceed 2100 mg/kg; in other embodiments, the total amount administered per course of treatment does not exceed 1400 mg/kg. For the aging-related disease or disorder to be treated, the optimum dose (daily or per course of treatment) may be different, and may also vary depending on the route of administration and treatment regimen.

Pharmaceutical compositions comprising senolytic agents can be formulated in a manner suitable for use in a delivery method by using techniques routinely practiced in the art. The composition may be in the form of a solid (e.g., tablet, capsule), semi-solid (e.g., gel), liquid, or gas (aerosol). In certain other particular embodiments, the senolytic agent (or pharmaceutical composition comprising the same) is administered as an intravenous bolus infusion. In certain embodiments, when the senolytic agent is delivered by infusion, the senolytic agent is delivered via a blood vessel into an organ or tissue comprising the senescent cells to be killed according to techniques routinely performed by those of skill in the medical arts.

Pharmaceutically acceptable Excipients are well known in the Pharmaceutical art and are described, for example, in Rowe et al, Handbook of Pharmaceutical Excipients: A Comprehensive Guide to Uses, Properties, and safety,5^thEd, 2006 and Remington The Science and Practice of Pharmacy (Gennaro, 21)^stMac k pub co, Easton, PA (2005)). Exemplary pharmaceutically acceptable excipients include sterile saline and phosphate buffered saline at physiological pH. Preservatives, stabilizers, dyes, buffers, and the like may be provided in the pharmaceutical compositions. In addition, antioxidants and suspending agents may also be used. In general, it is preferred that,the type of excipient is selected according to the mode of administration and the chemical composition of the active ingredient. Alternatively, the compositions described herein may be formulated as a lyophilizate. The compositions described herein may be lyophilized or otherwise formulated as a lyophilized product using one or more suitable excipient solutions for dissolving and/or diluting the pharmaceutical agents of the composition upon administration. In other embodiments, the agent is encapsulated within liposomes using techniques known and practiced in the art. In certain particular embodiments, the senolytic agent (e.g., ABT-263) is not formulated within a liposome for application to a stent for treating a highly, but not completely, occluded artery. The pharmaceutical compositions may be formulated for any suitable mode of administration described herein and in the art.

The pharmaceutical composition can be delivered to a subject in need thereof by any of several routes known to those skilled in the art. By way of non-limiting example, the composition can be delivered orally, intravenously, intraperitoneally, by infusion (e.g., bolus infusion), subcutaneously, enterally, rectally, intranasally, by inhalation, buccally, sublingually, intramuscularly, transdermally, intradermally, topically, intraocularly, vaginally, rectally, or by intracranial injection, or any combination thereof. In certain particular embodiments, administration of the dose as described above is via intravenous, intraperitoneal, direct access to the target tissue or organ, or via subcutaneous route. In certain embodiments, the method of delivery comprises a drug-coated or infiltrated scaffold, wherein the drug is a senolytic agent. Formulations suitable for such delivery methods are described in more detail herein.

In certain particular embodiments, the senolytic agent (which can be combined with at least one pharmaceutically acceptable excipient to form a pharmaceutical composition) is administered directly to a target tissue or organ comprising senescent cells that cause the disease or condition to manifest. In particular embodiments, in treating osteoarthritis, at least one senolytic agent is administered directly to the osteoarthritic joint (i.e., intra-articular) in a subject in need thereof-in other particular embodiments, the senolytic agent may be administered to the joint via a topical, transdermal, intradermal, or subcutaneous route. In certain other embodiments, provided herein are methods for treating cardiovascular diseases or disorders associated with atherosclerosis, such as atherosclerosis, by direct administration into an artery. In another particular embodiment, the senolytic agent (which may be combined with at least one pharmaceutically acceptable excipient to form a pharmaceutical composition) for use in treating a senescence-associated lung disease or disorder can be administered by inhalation, intranasally, by intubation, or intratracheally, to provide the senolytic agent, e.g., more directly, to the infected lung tissue. By way of another non-limiting example, the senolytic agent (or pharmaceutical composition comprising the senolytic agent) can be delivered directly to the eye by injection (e.g., intraocular or intravitreal) or by conjunctival application under the eyelid of a cream, ointment, gel, or eye drop. In more particular embodiments, the senolytic agent or pharmaceutical composition comprising the senolytic agent can be formulated as a delayed release (also referred to as sustained release, controlled release) composition or can be administered as an intravenous bolus infusion.

The pharmaceutical composition (e.g., for oral administration or for injection, infusion, subcutaneous delivery, intramuscular delivery, intraperitoneal delivery, or other methods) can be in the form of a liquid. For example, the liquid pharmaceutical composition may comprise one or more of the following: sterile diluents such as water, saline solution (preferably physiological saline), ringer's solution, isotonic sodium chloride, fixed oils as a solvent or suspending medium, polyethylene glycol, glycerol, propylene glycol or other solvents; an antibacterial agent; an antioxidant; a chelating agent; buffers and agents for regulating osmotic pressure such as sodium chloride or glucose. The parenteral compositions may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. The use of physiological saline is preferred, and injectable pharmaceutical compositions are preferably sterile. In another embodiment, to treat an ophthalmic condition or disease, the liquid pharmaceutical composition may be administered to the eye in the form of eye drops. The liquid pharmaceutical composition may be delivered orally.

For oral formulations, at least one senolytic agent as described herein can be used alone or in combination with suitable additives to prepare tablets, powders, granules or capsules, and if desired, in combination with diluents, buffers, wetting agents, preservatives, colorants and flavoring agents. The compound may be formulated with buffers to provide protection of the compound from the low pH of the gastric environment, and/or with enteric coatings. Senolytic agents included in a pharmaceutical composition can be formulated with a flavoring agent, for example, in a liquid, solid, or semi-solid formulation, and/or with an enteric coating for oral delivery.

The pharmaceutical composition comprising any of the senolytic agents described herein can be formulated for sustained release or slow release (also referred to as delayed release or controlled release). Such compositions may generally be prepared using well-known techniques and administered by, for example, oral, rectal, intradermal or subcutaneous implantation, or by implantation at the desired target site. Sustained release formulations may contain the compound dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane. Excipients used within such formulations are biocompatible and may also be biodegradable; preferably, the formulation provides a relatively constant level of active ingredient release. The amount of active agent included in the sustained release formulation depends on the site of implantation, the rate and desired length of release, and the nature of the condition, disease or disorder to be treated or prevented.

In certain embodiments, the pharmaceutical composition comprising the senolytic agent is formulated for transdermal, intradermal, or topical administration. The composition can be administered in the form of a powder/talc or other solid, liquid, spray, aerosol, ointment, foam, cream, gel, paste using a syringe, bandage, transdermal patch, insert, or syringe-like applicator. This is preferably in the form of a controlled or sustained release formulation for topical application or direct injection (intradermal or subcutaneous) to the skin in or near the area to be treated. The active component may also be delivered via iontophoresis. Preservatives can be used to prevent the growth of fungi and other microorganisms. Suitable preservatives include, but are not limited to, benzoic acid, butylparaben, ethylparaben, methylparaben, propylparaben, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, thimerosal, and combinations thereof.

The pharmaceutical composition comprising the senolytic agent can be formulated as an emulsion for topical administration. The emulsion comprises one liquid dispersed in a body of a second liquid. The emulsion may be an oil-in-water emulsion or a water-in-oil emulsion. Either or both of the oil and water phases may contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other excipients. The oil phase may contain other pharmaceutically acceptable oily excipients. Suitable surfactants include, but are not limited to, anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants. Compositions for topical application may also include at least one suitable suspending agent, antioxidant, chelating agent, emollient, or humectant.

For example, ointments and creams may be formulated with aqueous or oily bases by the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. The liquid spray may be delivered from the pressurized package via, for example, a specially shaped closure. Oil-in-water emulsions are also useful in compositions, patches, bandages and articles. These systems are semisolid emulsion, microemulsion or foam emulsion systems.

In some embodiments, the senolytic agent can be formulated with an oily base or ointment to form a semi-solid composition having a desired shape. In addition to senolytic agents, these semi-solid compositions may contain dissolved and/or suspended antimicrobials, preservatives, and/or buffering systems. The petrolatum component which may be included may be any paraffin wax having a viscosity ranging from mineral oil containing isobutylene, colloidal silica or stearate to paraffin wax. The absorbent matrix may be used with an oil-containing system. Additives may include cholesterol, lanolin (lanolin derivatives, beeswax, fatty alcohols, lanolin wax alcohols, low HLB (hydrophilic lipophilic balance) emulsifiers, and various ionic and nonionic surfactants, alone or in combination.

Controlled or sustained release transdermal or topical formulations can be achieved by the addition of time-release additives, such as polymeric structures, matrices, as are available in the art. For example, the composition can be applied by using a hot melt extruded article (e.g., a bioadhesive hot melt extruded film). The formulation may comprise a crosslinked polycarboxylic acid polymer formulation. The cross-linking agent may be present in an amount that provides sufficient adhesion to allow attachment of the system to the surface of the target epithelial or endothelial cell for a sufficient time to allow the desired release of the compound.

The insert, transdermal patch, bandage or article may comprise a mixture or coating of polymers that provides a constant rate of release of the active agent over an extended period of time. In some embodiments, the article, transdermal patch, or insert comprises a water-soluble pore former such as polyethylene glycol (PEG) that may be mixed with a non-water soluble polymer to increase the durability of the insert and prolong the release of the active ingredient.

Transdermal devices (inserts, patches, bandages) may also comprise water insoluble polymers. The rate controlling polymer may be useful for site administration, where a change in pH may be used to effect release. These rate controlling polymers can be applied using a continuous coating film during the process of spraying and drying with the active compound. In one embodiment, the coating formulation is used to coat a pellet containing an active ingredient that is compressed to form a solid biodegradable insert.

Polymeric formulations may also be used to provide controlled or sustained release. Bioadhesive polymers described in the art may be used. By way of example, the sustained release gel and the compound may be incorporated into a polymeric matrix, such as a hydrophobic polymeric matrix. Examples of polymer matrices include microparticles. The microparticles may be microspheres and the core may be a different material than the polymeric shell. Alternatively, the polymer may be cast as a sheet or film, a powder produced by grinding or other standard techniques, or a gel such as a hydrogel. The polymer may also be in the form of a coating or portion of a bandage, stent, catheter, vascular graft, or other device to facilitate the delivery of senolytic agents. The matrix may be formed by solvent evaporation, spray drying, solvent extraction, and other methods known to those skilled in the art.

In certain embodiments of the methods described herein for treating a cardiovascular disease associated with or caused by arteriosclerosis, one or more senolytic agents can be delivered directly into a blood vessel (e.g., artery) via a stent. In particular embodiments, the stent is used to deliver senolytic agents to an atherosclerotic vessel (artery). Stents are generally tubular metal devices that have a thin metal mesh-like architecture and which are inserted in a compressed form and then expanded at a target site. Stents are intended to provide long-term support to an expanded vessel. Several methods for preparing drug-coated and drug-embedded stents are described in the art. For example, senolytic agents can be incorporated into a polymer layer applied to a stent. A single type of polymer may be used and one or more layers of senolytic agent-permeated polymer may be applied to a bare metal stent to form a senolytic agent-coated stent. Senolytic agents may also be added to the pores of the metal scaffold itself, which may also be referred to herein as senolytic agent-permeated scaffolds or senolytic agent-embedded scaffolds. In certain particular embodiments, the senolytic agent can be formulated within a liposome and applied to a scaffold; in other particular embodiments, for example, when the senolytic agent is ABT-263, ABT-263 is not formulated in a liposome. Placement of the stent in the atherosclerotic artery is performed by one skilled in the medical arts. The senolytic agent coated or embedded stent not only expands the infected vessel (e.g., artery), but is also effective for one or more of (1) reducing the amount of plaque, (2) inhibiting plaque formation, and (3) increasing the stability of the plaque (e.g., by reducing the lipid content of the plaque; and/or causing an increase in the thickness of the fibrous cap), particularly for plaque proximal to the agent coated or agent embedded stent.

In a particular embodiment, the senolytic agent administered to a subject having an ocular senescence-associated disease or disorder can be delivered intraocularly or intravitreally. In other embodiments, the senolytic agent can be administered to the eye by applying the senolytic agent to the mucosa and tissue of the upper eyelid, the lower eyelid, or both via the conjunctival route. Any of these administrations may be bolus infused. In other particular embodiments, a pharmaceutical composition comprising any of the senolytic agents described herein can be formulated for sustained or slow release (which can also be referred to as delayed release or controlled release), which formulation is described in more detail herein. In certain embodiments, provided herein are compositions for treating or preventing (i.e., reducing the likelihood of occurrence; delaying onset or development; or inhibiting, delaying, slowing, or impeding progression or severity) an ocular disease, disorder, or condition (e.g., presbyopia, cataract, macular degeneration); methods for selectively killing senescent cells in an eye of a subject, and/or inducing collagen (such as type IV collagen) production in an eye of a subject in need thereof by directly administering to the eye at least one senolytic agent (which can be combined with at least one pharmaceutically acceptable excipient to form a pharmaceutical composition).

For pharmaceutical compositions comprising a nucleic acid molecule, the nucleic acid molecule can be present in a variety of delivery systems known to those of ordinary skill in the art, including nucleic acids and bacterial, viral, and mammalian expression systems, such as, for example, recombinant expression constructs as provided herein. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. For example, the DNA may also be referred to as "naked" as described in Ulmer et al, Science 259: 1745-. The uptake of naked DNA can be increased by coating the DNA onto biodegradable beads that are efficiently transported into the cell. Nucleic acid molecules can be delivered into cells according to any of several methods described in the art (see, e.g., Akhtar et al, Trends Cell Bio.2:139 (1992); Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar,1995, Maurer et al, mol. Membr. biol.16:129-40 (1999); Hofland et al, handbb. exp. Pharmacol.137:165-92 1999; Lee et al, ACS Symp. Ser.752:184-92 (2000); U.S. Pat. No. 6,395,713; International patent application publication No. WO 94/02595; Selbo et al, int. J. cancer 87:853-59 (2000); Selbo et al, Tumour biol.23: 3812; U.S. patent publication No. 2002-2001/0007666; 2003/077829).

Kits are provided having a unit dose (typically an oral or injectable dose) of one or more of the agents described herein. Such kits comprise a container containing the unit dose, an information package insert describing the use and concomitant benefits of the drug in treating the disease associated with aging cells, and optionally an instrument or device for delivering the composition.

Examples

Example 1

In vitro cellular assay for determining senolytic activity of Nutlin-3a

Foreskin fibroblast lines HCA2 and BJ, lung fibroblast line IMR90, and mouse embryonic fibroblasts were seeded in a six-well plate and senescence was induced by 10Gy of Ionizing Radiation (IR) or treatment with dorbixacin (Doxo) for 24 hr. The senescent phenotype was allowed to develop for at least 7 days, at which time point cell counts were performed to determine the baseline number of cells. Nutlin-3a treatment was then started for at least 9 days. The medium with the drug or medium only is refreshed as appropriate at least every three days. At the end of the assay period, cells were counted. Each case was seeded into three plate wells and counted independently. The initial cell count served as a control to determine the induction of senescence compared to the last day count without nutlin treatment. The initial senescent cell count served as a proxy (proxy) for determining Nutlin-3a toxicity. Figure 1 shows a schematic representation of the experimental design.

Foreskin fibroblast lines HCA2 and BJ, lung fibroblast line IMR90, and mouse embryonic fibroblasts were exposed to 10Gy of Ionizing Radiation (IR) to induce senescence. Seven days after irradiation, the cells were treated with Nutlin-3a (0, 2.5 μ M and 10 μ M) at different concentrations for 9 days, with the drug being refreshed at least every 3 days. Percent survival was calculated as [ Nutlin-3a day 9 cell count/Nutlin-3 a day initial cell count ]. The results are shown in FIGS. 2A-D, which show that Nutlin-3a reduces cell survival of senescent foreskin fibroblasts (HCA2 and BJ), lung fibroblasts (IMR90), and Mouse Embryonic Fibroblasts (MEF), indicating that Nutlin-3a is a senolytic agent.

Endothelial fibroblasts (HCA2) and aortic endothelial cells (Endo Aort) were treated with doxorubicin (250nM) for one day (24 hours) to induce senescence (see fig. 1). Eight days after doxorubicin treatment, Nutlin-3a treatment was initiated. HCA2 cells were exposed to Nutlin-3a for 9 days and aortic endothelial cells were exposed to Nutlin-3a for 11 days. The medium containing the compound or control medium is refreshed at least every 3 days. Percent survival was calculated as [ cell count on the last day of Nutlin-3a treatment/initial cell count on the first day of Nutlin-3a treatment ]. The results are shown in FIGS. 3A-B, which show that doxorubicin-induced senescent cells are sensitive to Nutlin-3A.

Non-senescent foreskin fibroblasts (HCA2), lung fibroblasts (IMR90) and Mouse Embryonic Fibroblasts (MEF) were treated with Nutlin-3a at different concentrations (0, 2.5. mu.M and 10. mu.M) for 9 days to evaluate Nutlin-3a toxicity. Cell counts were taken at the beginning (NS Start) and end of Nutlin-3a treatment. The difference between the count of cells not treated with Nutlin-3a at day 9 (NS 0) and the cell count determined at day 0 (NS start) reflects cell growth over a specified period of time. The results are shown in figures 4A-C, which show that Nutlin-3a treatment decreased proliferation but was non-toxic to non-senescent cells. Nutlin-3a treatment did not reduce the cell number below the initial level, indicating no toxicity. The reduction in apparent survival between NS 0 and NS 2.5 and between NS 0 and NS 10 reflects a reduction in cell growth. Without wishing to be bound by theory, Nutlin-3a may stabilize p53, thereby causing a resistance to cell cycle growth.

Non-senescent aortic endothelial (Endo Aort) cells and preadipocytes (Pread) were also treated with Nutlin-3a at different concentrations (0, 2.5 μ M and 10 μ M) for 11 days to evaluate Nutlin-3a toxicity, as described above. Cell counts were performed at the start (NS Start) and end (NS 0) of day 0 of Nutlin-3a treatment. The difference between the count at day 11 (NS 0) for cells not treated with Nutlin-3a and the count of cells from the beginning of NS reflects cell growth over the indicated time period. The results are shown in FIGS. 5A-B, demonstrating that Nutlin-3a treatment reduced proliferation but was non-toxic to non-senescent cells. Nutlin-3a treatment did not reduce cell number below the initial level, as observed by fibroblasts, indicating no toxicity. The reduction in apparent survival between NS 0 and NS 2.5 and between NS 0 and NS 10 reflects a reduction in cell growth.

Example 2

Nutlin-3a treatment of p16-3MR transgenic mice

The ability of Nutlin-3a to eliminate senescent cells in vivo was determined in transgenic p16-3MR mice (see, e.g., International application publication No. WO 2013/090645). A schematic representation of the experimental protocol is provided in fig. 6. The transgenic mice comprise p16 operably linked to a trimodal fusion protein^Ink4aThe promoter used to detect senescent cells and for selective clearance of senescent cells in these transgenic mice is shown in figure 7. Promoter p16 transcriptionally active in senescent cells but not in non-senescent cells^Ink4a(see, e.g., Wang et al, J. biol. chem.276:48655-61 (2001); Baker et al, Nature 479:232-36(2011)) are engineered into nucleic acid constructs. 3MR (trimorphous reporter) is a fusion protein containing the functional domains of synthetic renilla Luciferase (LUC), monomeric red fluorescent protein (mRFP) and truncated Herpes Simplex Virus (HSV) -1 thymidine kinase (tTK), which allows killing by more coxivir (GCV) (see, e.g., Ray et al, Cancer Res.64:1323-30 (2004)). The 3MRcDNA was inserted into exon 2 in frame with exon 16, resulting in a fusion protein containing the first 62 amino acids of p16, rather than the full-length wild-type p16 protein. Insertion of the 3MR cDNA also resulted in a stop codon at p19 in exon 2^ARFIn reading frame, thereby preventing full-length p19^ARFExpressed from BACs. P16^Ink4aA gene promoter (about 10 ten thousand base pairs) was introduced upstream of the nucleotide sequence encoding the trimodal reporter fusion protein. Alternatively, truncated p16 may be used^Ink4aPromoters (see, e.g., Baker et al, Nature, supra; International application publication No. WO 2012/177927; Wang et al, supra). Thus, 3MR watchUp to p16in senescent cells only^Ink4aAnd (3) a promoter driver. Detectable markers LUC and mRFP allow detection of senescent cells by bioluminescence and fluorescence, respectively. tTK allows selective killing of senescent cells by exposure to the prodrug Ganciclovir (GCV), which is converted by tTK into a cytotoxic moiety. Transgenic founder (founder) animals with a C57B16 background were established and bred using known procedures for introducing transgenes into animals (see, e.g., Baker et al, Nature 479:232-36 (2011)).

Female C57/BL6p16-3MR mice were randomized to either doxorubicin + Nutlin-3a treated group or doxorubicin only treated group (see FIG. 6). Senescence was induced by intraperitoneal administration of 10mg/kg of doxorubicin to mice ten days before Nutlin-3a administration (day-10). Nutlin-3a (25mg/kg) was administered intraperitoneally daily from day 10 to day 24 after treatment with dorbixin (group ═ 9 mice). Control mice (doxorubicin-treated) were injected with an equal volume of PBS (group ═ 3 mice). Luminescence imaging (Xenogen imaging system) was performed on day 0 (i.e., 10 days after doxorubicin treatment) as baseline (100% intensity) for each mouse.

Luminescence imaging of mice was performed on days 7, 14, 21, 28, and 35 after the start of Nutlin-3a treatment. The reduction in luminescence (L) is calculated as: l ═ imaging after Nutlin-3a treatment)/(baseline imaging)%. If L is 100% or more, the number of senescent cells is not decreased. If L is less than 100%, the number of senescent cells is reduced. Each mouse was calculated independently and the background was subtracted from each sample. The results are shown in figure 8, which shows that treatment with Nutlin-3a reduces the luminescence associated with doxorubicin-induced aging. Statistically, statistically significant reductions in luminescence were observed on days 14, 28 and 35 in Nutlin-3 a-treated animals.

Experiments were performed to determine the effect of Nutlin-3a treatment on the expression of senescence-associated genes. Groups of female C57/BL6p16-3MR were treated as described above. Three weeks after the end of Nutlin-3a treatment (day 35), doxorubicin-treated mice (control) (N ═ 3) and doxorubicin + Nutlin-3 a-treated mice (N ═ 6) were sacrificed. Skin and fat biopsies were collected for RNA extraction; fat biopsies were collected to detect senescence-associated β -galactosidase; and the lungs were rapidly frozen in cryoprotectant OCT media for cryostat sectioning.

Endogenous aging markers of RNA (p21, p16) were analyzed relative to actin mRNA (control for cDNA amount) using Roche Universal Probe Library for real-time PCR assay (p21, p16)^INK4a(p16) and p53) and SASP factors (mmp-3 and IL-6). The results are shown in FIGS. 9A-E, which show that Nutlin-3a treatment decreased the expression of SASP factor and senescence markers associated with doxorubicin-induced senescence. This value represents the fold induction of the corresponding mRNA relative to untreated control animals.

Frozen lung tissue was sectioned to 10 μ M thickness and stained with rabbit polyclonal primary antibody (Novus Biologicals, LLC) against γ H2AX, where γ H2AX is a marker of double strand breaks (DNA damage) in cells. Sections were then stained with ALEXA dye-labeled goat anti-rabbit secondary antibodies (Life Technologies) and counterstained with 4', 6-diamidino-2-phenylindole (DAPI) (Life Technologies). The number of positive cells was calculated using the ImageJ image processing program (National institutsof Health, see internet image j. nih. gov/ij/index. html) and expressed as a percentage of the total number of cells. The results are shown in FIGS. 10A-B, which show that nutlin-3A treatment reduced the number of cells with DNA damage induced by doxorubicin. Figure 10A shows a reduction in γ H2AX staining in doxorubicin + Nutlin-3a treated cells compared to doxorubicin alone. Figure 10B shows the reduction in the percentage of γ H2AX positive cells in doxorubicin + Nutlin-3a treated cells compared to doxorubicin alone.

At the time of collection, fat biopsies were immediately fixed in 4% formalin and then stained with a solution containing X-gal to detect the presence of senescence-associated β -galactosidase (β -gal). Fat biopsies were incubated overnight in X-gal solution at 37 ℃ and photographed the next day. Fat biopsies from untreated animals were used as negative Control (CTRL). The results are shown in figure 11, which shows that Nutlin-3a treatment reduced the senescence-associated β -gal intensity in fat biopsies from animals with doxorubicin-induced senescence, similar to the untreated negative control, compared to mice treated with doxorubicin alone.

Example 3

MDM2 inhibitors ablate senescent cells with established SASP

Primary human fibroblast (IMR90) cells were induced to senescence by applying 10Gy of irradiation. Seven days after irradiation (day 0), cells were treated with 10 μ M Nutlin-3a or vehicle (DMSO) for nine days (day 9). The drug or vehicle was renewed every three days. Drug/vehicle was removed on day 9 and cells were cultured for an additional three days (day 12). Cells were then fixed with 4% paraformaldehyde and stained by immunofluorescence with a specific anti-IL-6 antibody (R & D, AF-206-NA). Cells were counterstained with DAPI for nuclear visualization. The percentage of IL-6 positive cells is shown in FIG. 12A. An example of IL-6 positive cell immunofluorescence is shown in FIG. 12B. IL-6 positive cells were determined in a fair (unaided) manner using CellProfiler software. Three different cultures were evaluated. Non-senescent cells had no detectable cellular IL-6 production, while at day 9 after vehicle (DMSO) treatment (16 days post irradiation), senescent cells were about 8% positive. Nutlin-3a treatment reduced the percentage of IL-6 positive cells to a level below 5%. After three days, on day 12, Nutlin-3a was removed, and 19 days after irradiation, IL-6 positive cells in the vehicle control were about 9%, while Nutlin3a treated cells were less than 1% IL-6 positive.

In another experiment, IMR90 cells were induced to senescence by irradiation (10 Gy). Seven days after irradiation, cells were treated with 10. mu.M Nutlin-3a or vehicle (DMSO) for nine days (day 9).

IL-6 levels in senescent cells are 10-40 times higher than levels in non-senescent cells. Nutlin-3 a-treated senescent cells had IL-6 levels as low as 5-9 fold compared to DMSO-treated cells. Cells that survived Nutlin-3a treatment had lower IL-6 secretion and, by extrapolation, had lower SASP, indicating that Nutlin-3a preferentially killed senescent cells with well established SASP.

Example 4

MDM2 inhibitors remove senescent cells with established SASP:

SASP factor expression

Primary human fibroblast (IMR90) cells were induced to senescence by applying 10Gy of irradiation. Seven days after irradiation (day 0), cells were treated with 10 μ M Nutlin-3a or vehicle (DMSO) for nine days (day 9). The drug or vehicle was renewed every three days. The drug/vehicle was removed on day 9 and the cells were cultured for an additional three days in medium without drug or DMSO (day 12). Cells were then collected, mRNA was extracted and cDNA was prepared. Quantitative pcr (qpcr) was then performed to detect the expression of the different genes. Cells were also harvested on day 12 after the drug/vehicle had been removed for 3 days. Data are presented as the average of three samples. Data were normalized to actin and shown as a ratio to non-senescent cells. The data are presented in FIGS. 13A-13F.

Levels of p21 were up to about 10 fold in senescent cells, and p21 levels were higher (about 90 fold) when the cells were treated with Nutlin-3 a. Nutlin-3a stabilizes p53, while p53 is a transcription factor that activates the expression of the cell cycle-dependent kinase inhibitor p 21. On day 12, levels of p21 in DMSO-treated cells were comparable to those on day 9, which was also comparable to those in Nutlin-3 a-treated cells on day 12. These data indicate that the acute effect of Nutlin-3a on the cells was abolished three days after the removal of the drug exposure. The level of another senescence marker, P16, was increased in irradiated cells but not changed in the presence of Nutlin-3 a. Three days after drug removal (day 12), a decrease in p16 levels was observed. The level of IL-1a, a regulator of SASP, was reduced only after Nutlin-3a removal. CXCL-1, IL-6 and IL-8 are three other SASP factors. When Nutlin-3a is present, the levels of all three factors are reduced and remain lower after drug removal. These data show that cells surviving Nutlin-3a treatment have a lower level of p16, indicating that Nutlin-3a preferentially kills cells that are high p16 expressors. Similarly, the reduction of SASP factor in surviving cells also indicates that Nutlin-3a preferentially kills cells with higher SASP.

Example 5

MDM2 inhibitor abrogates senescent cells with an increased DNA damage response

Primary human fibroblast (IMR90) cells were induced to senescence by applying 10Gy of irradiation. Seven days after irradiation (day 0), cells were treated with 10 μ M Nutlin-3a or vehicle (DMSO) for nine days (day 9). The drug or vehicle was renewed every three days. The drug/vehicle was removed on day 9 and the cells were cultured in medium without drug or DMSO for an additional six days with medium changed every three days. Cells were collected at day 0 (non-senescent cells), day 9, day 12, and day 15, and proteins were extracted and processed for immunoblotting (Western blotting). Processing two samples at each time point; the results for one sample are provided in fig. 14.

The data show that the kinase ATM is less phosphorylated in cells that have been treated with Nutlin-3a, even when the drug has been removed (see pATM S1981). Similarly, the phosphorylation level of substrate H2AX of ATM was reduced after Nutlin3A treatment and also after drug removal (see γ H2 AX). In senescent cells, IkBa is degraded when the NF-kB pathway is activated, which produces SASP. The data show that after drug removal, the level of IkBa in Nutlin-3a treated cells approaches that of non-senescent cells. In the Nutlin-3a treated samples, the level of each of MDM2, p53, and p21 was elevated and decreased when the drug was removed.

These data also support that Nutlin-3a preferentially kills cells with higher SASP. Furthermore, because of the lower level of activated ATM in the surviving cells after drug treatment, these data indicate that the aged cells activated by DNA damage response are Nutlin-3a sensitive cells.

Example 6

Selective toxicity of ABT-263 to senescent cells using a cytometric assay

To determine whether ABT-263 is selectively toxic to senescent cells compared to non-senescent cells, a cell count assay was used to determine cell survival after treatment with ABT-263. A general timeline and procedure for a cytometric assay is shown in fig. 15. IMR90 cells (human primary lung fibroblasts (IMR90) (IMR-90(CCL-186TM, Mannassas, Virginia)) were seeded in six well plates and senescence was induced by 10Gy of Ionizing Radiation (IR) (day 0.) every 3 days medium was refreshed allowing the senescent phenotype to develop for 7 days, cell counting was performed at this time point to determine the baseline number of cells.

FIG. 16 shows the effect of ABT-263 on non-senescent cells measured as the percentage of viable cells after 24 hours. Addition of ABT-263 to non-senescent cells (middle bar) did not reduce cell growth below the initial level (left-most bar), indicating that there was no toxicity in non-senescent cells. ABT-263 untreated cells are shown on the far right as a control.

FIG. 17 shows the effect of ABT-263 on senescent cells measured as the percentage of viable cells after 24 hours. Addition of ABT-263 to senescent cells (middle bar) reduced cell growth to a number of cells below the initial level (left-most bar). ABT-263 treated cells had a cell count of 28% prior to ABT-263 treatment. ABT-263 untreated cells are shown on the far right as a control.

Example 7

Use ofSelective toxicity of ABT-263 to senescent cells as determined by cell viability

To determine whether ABT-263 is selectively toxic to senescent cells compared to non-senescent cells, a cell viability assay was used to evaluate cell survival after treatment with ABT-263. A general timeline and procedure for a cytometric assay is shown in fig. 18. IMR90 cells (human primary lung fibroblasts (IMR90) (IMR-90(CCL-186TM, Mannassas, Virginia)) were seeded in six well plates and senescence was induced by 10Gy of Ionizing Radiation (IR) (day 0.) medium was refreshed every 3 days allowing the senescent phenotype to develop for 7 days, cell counting at this time point to determine the baseline number of cells, followed by seeding into 96 well plates at day 8, senescent cells (irradiated) and non-senescent cells (non-irradiated cells) were exposed to successive dilutions of ABT-263 for 3 days ABT-263 concentrations ranged from 0.5nM to 3 μ M.

After three days of treatment (day 11), commercially available products were used(CTG) luminescence Cell Viability Assay (Promega Corporation, Madison, Wisconsin) cells were analyzed for Cell survival. The assay determines the number of viable cells in the culture medium based on the quantification of ATP present, which is an indicator of metabolically active cells.

FIG. 19 shows the IC50 curve for ABT-263 in senescent and non-senescent cells. The IC50 curve is a graph of the percent cell survival after ABT-263 treatment as determined by cell viability assay. The figure shows the effect of ABT-263 at various concentration levels on cell survival. The IC50 of ABT-263 to non-senescent cells was 2.4. mu.M, compared to 140nM IC50 to senescent cells, demonstrating the selective toxicity of ABT-263 to senescent cells. An in vitro theoretical therapeutic index of 17 was observed.

Example 8

Evaluation of Selective toxicity of ABT-263 against senescent cells of various cell types

The procedure of example 7 was repeated for other cell lines. Cell lines included primary renal cortical cells, ATCC Cat # PCS-400-AscoA 011 (FIG. 20), HCA2 foreskin fibroblasts (FIG. 21), primary small airway epithelial cells, ATCC Cat # PCS-301-AscoA 010 (Lung) (FIG. 22), human confluent preadipocytes (Pread) from patients (FIG. 23), Mouse Embryonic Fibroblasts (MEF) extracted from C57Bl6 mice (FIG. 24), primary coronary smooth muscle, ATCC Cat # PCS-100-AscoA 021(Smth Mscl) (FIG. 25).

The experiments performed in these other cell lines were performed essentially as described in example 7. As shown in FIG. 20, the IC50 of ABT-263 is 430nM for non-senescent cells compared to 25nM for senescent cells, demonstrating the selective toxicity of ABT-263 for senescent cells in renal epithelial cells.

As shown in FIG. 21, the IC50 of ABT-263 to non-senescent cells was non-toxic up to 3 μ M, compared to the IC50 to senescent cells at 410nM, confirming the selective toxicity of ABT-263 to senescent cells in HCA2 cells.

Example 9

Evaluation of Selective toxicity of ABT-263 and other Bcl-2 inhibitors on senescent human Primary Lung fibroblasts

To determine whether other Bcl-2 inhibitors exhibited selective toxicity to senescent cells over non-senescent cells, cells were treated with ABT-199 (selockem Cat # S8048, Houston, TX) or obatoclatx (selockemcat # S1057). ABT-199 and Obatoclax are known Bcl-2 inhibitors.

The experiments performed to evaluate the effect of these other Bcl-2 inhibitors were performed essentially as described in example 7. Cells were exposed to ABT-199 at serial dilution concentrations ranging from 15nM to 100 μ M (fig. 26 and 27). Cells were exposed to Obatoclax at concentrations ranging from 1.4nM to 9 μ M (fig. 28).

As shown in FIGS. 26-27, ABT-199 had IC50 values of 6 μ M-15.8 μ M in non-senescent cells compared to IC50 of 6.9 μ M-12.4 μ M in senescent cells. As shown in figure 28, obatoclatax had an IC50 value of 75nM in non-senescent cells compared to an IC50 of 125nM in senescent cells. FIGS. 26-28 demonstrate that ABT-199 and Obatoclax do not have the ability to selectively target senescent cells relative to non-senescent cells.

Compounds specific to Bcl-2A1 also did not selectively kill senescent cells. IMR90 cells were induced to senescence by irradiation as described in example 7. The irradiated IMR90 cells and non-senescent IMR90 cells were then exposed to a compound called ML214, which is a Bcl-2a1 specific inhibitor. The level of killing of senescent cells is comparable to the level of killing of non-senescent cells.

Example 10

Akt inhibitor MK-2206 alone and its selective toxicity in combination with ABT-263 against senescent cells

The effect of combining ABT-263 with Akt inhibitor MK-2206 was tested for selective toxicity in IMR90 cells in senescent cells compared to non-senescent cells. The procedure of example 7 was repeated, but with exposure to 10nM MK-2206(Selleckem, Cat # S1078) in addition to serial dilutions of ABT-263.

FIG. 29A shows a dose-dependent plot of ABT-263 treatment in combination with 10nM MK-2206 on senescent and non-senescent cells. ABT-263+ MK-2206 treated senescent cells had IC50 values of 0.083 μ M, while ABT-263+ MK-2206 cells in non-senescent cells had IC50 values >3 μ M, resulting in a selectivity index for senescent cells > 36.

The senescence-clearing effect of MK-2206 alone was determined by exposing senescent IMR90 cells and non-senescent IMR90 cells to serial dilutions of MK-2206 (see the procedure in example 7). The percent survival was determined and the results are shown in figure 29B.

Example 11

Animal studies for determining senolytic effects of ABT-263 in mice the senolytic effects of senolytic agents such as ABT-263 can be evaluated in animal models of senescence. Examples of such animal studies are described herein. Aging of animals can be induced by administration of doxorubicin followed by treatment with an aging scavenger. On day 35, mice were sacrificed and fat and skin were collected for RNA analysis, while lungs were collected and snap frozen for analysis by immuno-microscopy. RNA was analyzed for expression of SASP factors (mmp3, IL-6) and senescence markers (p21, p16, and p 53). DNA damage markers (γ H2AX) were analyzed for frozen lung tissue.

The mice to be tested contained a transgenic insert of p16-3 MR. 3MR (trimodal reporter) is a fusion protein containing the functional domains of synthetic Renilla Luciferase (LUC), monomeric Red fluorescent protein (mRFP) and truncated Herpes Simplex Virus (HSV) -1 thymidine kinase (tTK), which allows killing by Ganciclovir (GCV). The 3MR cDNA was inserted in exon 2 in frame with p16, resulting in a fusion protein containing the first 62 amino acids of p16, but not including the full length wild type p16 protein. 3 insertion of MR cDNA also p19 in exon 2^ARFIntroducing stop code into reading frameAnd (4) adding the active ingredients.

The effect of ABT-263 was analyzed by the decrease in luminescence intensity. Female C57/Bl6p16-3MR mice were treated with doxorubicin. Luminescence was measured after 10 days and used as baseline (100% intensity) for each mouse. ABT-263 was administered intraperitoneally daily from day 10 to day 24 after doxorubicin treatment. Luminescence was then measured on days 7, 14, 21, 28, 35 after ABT-263 treatment and the final value was calculated as% of baseline. Control animals (DOXO) were injected with an equal volume of PBS.

mRNA levels of endogenous mmp-3, IL-6, p21, p16, and p53 in skin and fat from animals treated with doxorubicin alone (DOXO) or doxorubicin plus ABT-263 were plotted. This value represents the fold induction of a particular mRNA compared to untreated control animals.

Immunofluorescence microscopy of lung sections from doxorubicin-treated (DOXO) and doxorubicin and ABT-263-treated animals can be performed by binding to rabbit polyclonal primary antibody specific for γ H2AX, followed by incubation with goat anti-rabbit secondary antibody, and then counterstaining with DAPI. The percentage of positive cells from immunofluorescence microscopy was calculated and can be expressed as a percentage of the total number of cells. Data can be obtained from doxorubicin-treated mice (Doxo) and doxorubicin + ABT-263 treated mice.

ABT-263 can be analyzed for a decrease in senescence-associated (SA) β -galactosidase (β -gal) intensity from fat biopsies from animals first treated with doxorubicin. Female C57/BL6p16-3MR mice were treated with doxorubicin. A portion of doxorubicin-treated animals received ABT-263 or pbs (doxo) daily on days 10 to 24 after doxorubicin treatment. Three weeks after ABT-263 treatment, mice were sacrificed and fat biopsies were immediately fixed and stained with a solution containing X-Gal. Untreated animals were used as negative Controls (CTRL).

Example 12

In vitro cellular assay for determining senolytic activity of WEHI-539

Lung fibroblast cell line IMR90 (human primary lung fibroblasts, CCL-186TM, Manassas, Virginia) and kidney cell line (primary renal cortical cells, ATCC accession number PCS-400-011) were seeded in six-well plates and senescence was induced by 10Gy of Ionizing Radiation (IR). Allowing the senescent phenotype to develop for at least 7 days.

After the senescent phenotype developed, cells were re-seeded into 96-well plates and senescent cells (irradiated) and non-senescent cells (non-irradiated) were exposed to a three-fold serial dilution of WEHI-539 for a period of 3 days. WEHI-539 concentrations ranged from 0.0075 μ M to 15 μ M. After three days, use is made of a commercially available productCell survival was determined by luminescennt Cell Viabi lite Assay (Promega Corporation, Madison, Wisconsin). The assay determines the number of viable cells in the culture medium based on the quantification of ATP present, which is an indicator of metabolically active cells. Fig. 30 shows IMR90 cell survival (see fig. 30A) and kidney cell survival (see fig. 30B).

Example 13

WEHI-539 treatment of p16-3MR transgenic mice

This example describes an animal model useful for determining the ability of senolytic agents to selectively kill senescent cells in vivo. The ability of WEHI-539 or another senolytic agent to eliminate senescent cells in vivo was determined in transgenic p16-3MR mice (see, e.g., International application publication No. WO 2013/090645). The experiment was performed in a similar manner to the procedure description in the diagram provided in fig. 6. The transgenic mice comprise p16 operably linked to a trimodal fusion protein^Ink4aThe promoter used to detect senescent cells and for selective clearance of senescent cells in these transgenic mice is shown in figure 7. Promoter p16 transcriptionally active in senescent cells but not in non-senescent cells^Ink4a(see, e.g., Wang et al, J.biol.chem.276: 48655-61 (2001); Baker et al, Nature 479:232-36(2011)) have been engineered into nucleic acid constructs. 3MR (trimodal reporter) is a fusion protein containing the functional domains of synthetic Renilla Luciferase (LUC), monomeric Red fluorescent protein (mRFP) and truncated Herpes Simplex Virus (HSV) -1 thymidine kinase (tTK), which allows passage through more coxibLovir (GCV) kill (see, e.g., Ray et al, Cancer Res.64:1323-30 (2004)). The 3MR cDNA was inserted into exon 2 in frame with p16, resulting in a fusion protein containing the first 62 amino acids of p16, rather than the full-length wild-type p16 protein. Insertion of the 3MR cDNA also resulted in a stop codon at p19 of exon 2^ARFIn reading frame, thereby preventing full-length p19^ARFExpressed from BACs. P16^Ink4aA gene promoter (about 10 ten thousand base pairs) was introduced upstream of the nucleotide sequence encoding the trimodal reporter fusion protein. Alternatively, truncated p16 may be used^Ink4aPromoters (see, e.g., Baker et al, Nature, supra; International application publication No. WO 2012/177927; Wang et al, supra). Thus, the expression of 3MR was only from p16in senescent cells^Ink4aAnd (3) a promoter driver. Detectable markers LUC and mRFP allow detection of senescent cells by bioluminescence and fluorescence, respectively. tTK allows selective killing of senescent cells by exposure to the prodrug Ganciclovir (GCV), which is converted to a cytotoxic moiety by tTK. Transgenic founder animals with a C57B16 background were established and bred using known procedures for introducing transgenes into animals (see, e.g., Baker et al, Nature 479:232-36 (2011)).

To determine the senolytic activity of agents such as WEHI-539, female C57/BL6p16-3MR mice were randomly divided into doxorubicin + WEHI-539-treated groups or doxorubicin-only treated groups. Senescence was induced by intraperitoneal administration of dorbixin at 10mg/kg to mice ten days before administration of WEHI-539 (day-10). WEHI-539 (group of 9 mice) was administered intraperitoneally daily from day 10 to day 24 after doxorubicin treatment. Control mice (doxorubicin-treated) were injected with an equal volume of PBS (group ═ 3 mice). Luminescence imaging (Xenogen imaging system) was performed on day 0 (i.e., 10 days after dorbixacin treatment) as baseline (100% intensity) for each mouse.

Luminescence imaging of mice was performed on days 7, 14, 21, 28, and 35 after the start of WEHI-539 treatment. The reduction in luminescence (L) is calculated as: l ═ imaging after WEHI-539 treatment)/(baseline imaging)%. If L is 100% or more, the number of senescent cells is not decreased. If L is less than 100%, the number of senescent cells is reduced. Each mouse was calculated independently and the background was subtracted from each sample.

Experiments were performed to determine the effect of WEHI-539 treatment on the expression of senescence-associated genes. Groups of female C57/BL6p16-3MR were treated as described above. Three weeks after the completion of the WEHI-539 treatment (day 35), doxorubicin-treated mice (control) (N ═ 3) and doxorubicin + WEHI-539-treated mice (N ═ 6) were sacrificed. Skin and fat biopsies were collected for RNA extraction; fat biopsies were collected to detect senescence-associated β -galactosidase; and lungs were snap frozen in cryoprotectant OCT medium for cryosectioning.

Endogenous aging markers of RNA (e.g., p21, p16) were analyzed relative to actin mRNA (control for cDNA amount) using Roche Universal Probe Library for real-time PCR assays^INK4a(p16) and p53) and SASP factors (e.g., mmp-3 and IL-6).

Frozen lung tissue was sectioned to 10 μ M thickness and stained with rabbit polyclonal primary antibody (Novus Biologicals, LLC) against γ H2AX, where γ H2AX is a marker of double strand breaks (DNA damage) in cells. Then using ALEXA for the sectionDye-labeled goat anti-rabbit secondary antibodies (Life Technologies) were stained and counterstained with 4', 6-diamidino-2-phenylindole (DAPI) (Life Technologies). The number of positive cells was calculated using the ImageJ image processing program (national institutes of Health, see internet ImageJ. nih. gov/ij/index. html) and expressed as a percentage of the total number of cells.

At the time of collection, fat biopsies were immediately fixed in 4% formalin and then stained with a solution containing X-gal to detect the presence of senescence-associated β -galactosidase (β -gal). Fat biopsies were incubated overnight in X-gal solution at 37 ℃ and photographed the next day. Fat biopsies from untreated animals were used as negative Control (CTRL).

Example 14

Ability of BCL-XL inhibitors to remove senescent cells with established SASP

This example describes a method for determining the effect of senolytic agents to kill senescent cells with established SASP. Primary human fibroblast (IMR90) cells were induced to senescence by applying 10Gy of irradiation. Seven days after irradiation (day 0), cells were treated with 10 μ M BCL-XL inhibitor (e.g., WEHI-539) or BCL-2/BCL-XL inhibitor or vehicle (DMSO) for nine days (day 9). The drug or vehicle was renewed every three days. Drug/vehicle was removed on day 9 and cells were cultured for an additional three days (day 12). Cells were then fixed with 4% paraformaldehyde and stained by immunofluorescence with a specific anti-IL-6 antibody (R & D, AF-206-NA). Cells were counterstained with DAPI for nuclear visualization. IL-6 positive cells were determined in a fair manner using CellProfiler software.

In another experiment, IMR90 cells were induced to senescence by irradiation (10 Gy). Seven days after irradiation, cells were treated with senolytic agents (e.g., BCL-XL inhibitors (e.g., WEHI-539) or BCL-2/BCL-XL inhibitors; MDM2 inhibitors; Akt inhibitors) or vehicle (DMSO) for nine days (day 9). The drug or vehicle was renewed every three days. Drug/vehicle was removed on day 9 and cells were cultured for an additional six days. Conditioned media from treated cells were collected and IL-6 measurements were performed by ELISA (Perkin Elmer, AL 223F). IL-6 levels in the medium were determined by ELISA using the kit according to the manufacturer's instructions (AL223F, Perkin Elmer). Cells were fixed with 4% paraformaldehyde and stained by immunofluorescence with a specific anti-IL-6 antibody (R & D, AF-206-NA). IL-6 levels determined by ELISA were normalized to the number of cells in each well.

Example 15

Capacity of senolytic agents to remove senescent cells with established SASP: SASP factor expression

This example describes a method for determining the effect of senolytic agents on the expression of a SASP factor. Primary human fibroblast (IMR90) cells were induced to senescence by applying 10Gy of irradiation. Seven days after irradiation (day 0), cells were treated with senolytic agents (e.g., BCL-XL inhibitors (e.g., WEHI-539) or BCL-2/BCL-XL inhibitors; MDM2 inhibitors; Akt inhibitors) or vehicle (DMSO) for nine days. The drug or vehicle was renewed every three days. After removal of drug/vehicle before evaluating SASP expression on day 9, cells were cultured in media without drug or DMSO for an additional three days. Cells were then harvested, mRNA extracted and cDNA prepared. Quantitative pcr (qpcr) was then performed to detect the expression of the different genes. Cells were also harvested on day 12 after the drug/vehicle had been removed for three days. Data were normalized to actin and shown in proportion to non-senescent cells.

Example 16

Senolytic agents' ability to remove senescent cells with increased DNA damage response

This example describes a method for determining the effect of senolytic agents in selectively killing senescent cells that have an increased DNA damage response. Primary human fibroblast (IMR90) cells were induced to senescence by applying 10Gy of irradiation. Seven days after irradiation (day 0), cells are treated with senolytic agents (e.g., a BCL-XL inhibitor (e.g., WEHI-539) or a BCL-2/BCL-XL inhibitor, a MDM2 inhibitor; an Akt inhibitor) or vehicle (DMSO) for nine days (day 9). The drug or vehicle was refreshed every three days. The drug/vehicle was removed on day 9 and the cells were cultured in medium without drug or DMSO for an additional six days with medium changed every three days. Cells were collected at day 0 (non-senescent cells), day 9, day 12 and day 15, and proteins were extracted and processed for immunoblotting (Western blot). Two samples were processed at each time point.

Example 17

BCL-XL Selective inhibitors kill senescent cells via apoptosis

As described in example 12, the lung fibroblast cell line IMR90 (human primary lung fibroblasts,CCL-186TM, Manassas, Virginia) was seeded in six well plates and senescence was induced by 10Gy of Ionizing Radiation (IR). After senescence was established, cells were re-seeded in 96-well plates. The pan-caspase inhibitor Q-VD-OPh (20. mu.M) was added to senescent cells (spokes)Illuminated) (IMR90Sen (IR)) and added to wells containing non-senescent cells (unirradiated cells) (IMR90 NS). Four hours later, senescent cells and non-senescent cells were exposed to 1.67 or 5 μ M WEHI-539 for 3 days, respectively. At the end of the assay period, cells were counted. Each case was seeded into three plate wells and counted independently. The initial cell count served as a control to determine the induction of senescence compared to the last day count without WEHI-539 treatment. The initial non-senescent cell count served as a proxy (proxy) for determining WEHI-539 toxicity. Using commercially availableCell survival was determined by luminescennt Cell Viability Assay (Promega Corporation, Madison, Wisconsin). The assay determines the number of viable cells in the culture medium based on the quantification of ATP present, which is an indicator of metabolically active cells. Figure 31 (left) is a graphical representation of the selective killing of senescent cells by WEHI-539 (see example 12) and shows the concentrations of WEHI-539 used in this experiment. The percentage of surviving senescent cells increased in the presence of pan-caspase inhibitors (figure 31, right).

Example 18

Efficient killing of senescent cells by inhibition of BCL-XL

This example demonstrates that BCL-XL is a member of the BCL-2 anti-apoptotic family important for apoptosis of senescent cells. Short hairpin rnas (shrnas) comprising sequences specific for BCL-2, BCL-XL (also known as BCL2L1), and BCL-w (also known as BCL2L2) were prepared and introduced into lentiviral vectors. Four different shRNAs for each of BCL-XL and BCL-w and three shRNAs for BCL-2 were synthesized by the Broad Institute of MIT and Harvard (Cambridge, MA). Lentiviral vectors containing each corresponding shRNA were purchased from Sigma Aldrich (st. louis, MO). shRNA sequences and target sequences are provided in the table below. The nucleotide sequence of each protein can be readily obtained from public databases (see, e.g., Bcl-xL (Bcl 2-like 1(Bcl2L1)) at GenBank NM _001191.2 and NM _138578.1, Bcl-w (Bcl 2-like 2(Bcl2L2)) at GenBank NM _004050.3, and Bcl-2 (B-cell CLL/lymphoma 2(Bcl2)) at NM _000633.2, NM _ 000657).

Triplicate samples of senescent and non-senescent cells were transduced with each of the different lentiviral vectors as well as two control vectors according to methods practiced in the art. Control samples contained senescent cells and non-senescent cells that were not transduced with lentiviruses (NTs). IMR90 cells were induced to senescence by exposure to 10Gy of Ionizing Radiation (IR) as described in example 12. After the senescence phenotype developed, cells were re-seeded in 96-well plates and shRNA was added. After 24hr, the shRNA was removed and the medium was refreshed. The medium was renewed again after 3 days. After the last medium renewal (6 days after shRNA removal), the culture was performedLuminescab Cel l Viabi lite Assay measures survival.

Table: shRNA sequence

Then, for each shRNA tested, the survival of senescent and non-senescent cells was determined in triplicate. The shrnas listed in order in the table are presented from left to right in the figure. The second and third shRNA sequences specific for BCL-2 are identical. The ratio of senescent cell survival to non-senescent cell survival for each shRNA is presented in figure 32. A ratio of 1.0 indicates that the survival ratio of senescent cells is not different compared to non-senescent cells. The introduction of three of the four Bcl-XL specific shRNA molecules into senescent cells resulted in significant senescent cell death as compared to senescent cells into which Bcl-w or Bcl-2 specific shRNA was introduced. The data show that BCL-XL expression is important for the survival of senescent cells.

Example 19

Efficient killing of senescent cells by inhibition of Bcl-2 anti-apoptotic protein family members

To determine whether other Bcl-2/Bcl-xL/Bcl-w inhibitors are selectively toxic to senescent cells compared to non-senescent cells, a cell viability assay was used to evaluate cell survival following treatment with ABT-737. A general timeline and procedure for cell count assays is shown in fig. 18 and described in example 7. IMR90 cells (human primary lung fibroblasts) were seeded in six-well plates and senescence was induced by 10Gy of Ionizing Radiation (IR) (day 0). The medium was refreshed every 3 days. The senescent phenotype was allowed to develop for 7 days, at which time point cell counts were performed to determine the baseline number of cells, which were then seeded into 96-well plates. On day 8, senescent cells (irradiated) and non-senescent cells (non-irradiated cells) were exposed to serial dilutions of ABT-737 for a period of 3 days. ABT-737 concentrations were serially diluted from 50. mu.M. Each case was inoculated in triplicate.

After three days of treatment (day 11), use was made(CTG) luminescence Cell Viability Assay cells were analyzed for Cell survival. The assay determines the number of viable cells in the culture medium based on the quantification of ATP present, which is an indicator of metabolically active cells.

FIG. 33 shows the IC50 curves for ABT-737 in senescent and non-senescent cells. The IC50 curve is a graph of the percent cell survival after ABT-737 treatment as determined by cell viability assays. The figure shows the effect of ABT-737 at various concentration levels on cell survival.

Example 20

BCL-2/BCL-xL/BCL-w inhibitors kill senescent cells via apoptosis

An experiment as described in example 17 was performed to determine whether one or more other inhibitors of BCL-2 anti-apoptotic family members kill senescent cells by apoptosis. As described in example 12, the lung fibroblast cell line IMR90 (human primary lung fibroblasts,CCL-186TM, Manassas,Virginia) were inoculated in six-well plates and senescence was induced by 10Gy of Ionizing Radiation (IR). After senescence was established, cells were re-seeded in 96-well plates. The pancaspase inhibitor Q-VD-OPh (20 μ M) was added to the wells of senescent cells (irradiated) (IMR90Sen (IR)) and to the wells containing non-senescent cells (non-irradiated cells) (IMR90 NS). After four hours, senescent and non-senescent cells were exposed to 0.33 or 1 μ M ABT-263(Navitoclax), respectively, for 3 days. At the end of the assay period, cells were counted. Each case was seeded into three plate wells and counted independently. The initial cell count served as a control to determine the induction of senescence compared to the last day count without ABT-263 treatment. The initial non-senescent cell count serves as a proxy (proxy) for determining the toxicity of ABT-263. Use ofCell survival was determined by luminescennt Cell Viability Assay (Promega Corporation, Madison, Wisconsin). The assay determines the number of viable cells in the culture medium based on the quantification of ATP present, which is an indicator of metabolically active cells. FIG. 34 (upper panel) is a graphical representation of the selective killing of senescent cells by ABT-263 and shows the concentration of ABT-263 used in this experiment. In the presence of pan-caspase inhibitors, the percentage of senescent cells that were alive was increased (fig. 34, lower panel).

Example 21

Effect of senescent cell depletion in animal models of osteoarthritis

Tables and graphical representations of two osteoarthritis mouse model study designs are presented in figures 35 and 36, respectively. Two treatment studies were designed to determine the effect of senescent cell depletion in animal models of osteoarthritis.

A parallel study was performed. One study investigated the effect of Ganciclovir (GCV) on eliminating senescent cells in 3MR mice. Mice were subjected to surgery to remove the anterior cruciate ligament of one hindlimb, thereby inducing osteoarthritis in the joints of that lower limb. The surgical joints of 3MR mice received 2.5 μ g of GCV daily by intra-articular injection for 5 days during the 2 nd week post-surgery, with the 2 nd treatment at the 4 th week post-surgery (2.5 μ g of GCV daily for 5 days). At the end of 4 weeks post-surgery, mice were monitored for the presence of senescent cells in the surgical joints, evaluated for their function, monitored for markers of inflammation, and evaluated histologically.

In a parallel study, C57BL/6J mice were subjected to surgery to resect the anterior cruciate ligament of one hind limb, thereby inducing osteoarthritis in the joints of that lower limb. Mice were treated with 5.8 μ g of Nutlin-3A (n ═ 7) per operated knee by intra-articular injection for 2 weeks every other day on weeks 3 and 4 after surgery. At the end of 4 weeks post-surgery, the mouse joints were monitored for the presence of senescent cells, their function assessed, markers of inflammation monitored, and histologically assessed.

Two mouse control groups were included in the study performed: one group comprised C57BL/6J or 3MR mice that had undergone sham surgery (i.e., following surgical procedures except for resection of the ACL) and intra-articular vehicle injection in parallel with the GCV-treated group (n ═ 3); and one group comprised C57BL/6J or 3MR mice that had undergone ACL surgery and received intra-articular vehicle injections in parallel with the GCV-treated group (n ═ 5).

RNA from surgical joints of mice (from Nutlin-3A-treated mice) was analyzed for SASP factor (mmp3, IL-6) and senescence marker (p16) expression. qRT-PCR was performed to detect mRNA levels. Treatment with Nutlin-3A cleared senescent cells from the joint as shown in FIGS. 37A-C. RNA from a surgical joint of a mouse was also analyzed for expression of type 2 collagen, and compared with expression of actin as a control. As shown in figure 38, treatment with Nutlin-3A drives collagen production in mice that have undergone osteoarthritis surgery as compared to untreated mice.

At 4 weeks post-surgery, the weight-bearing test was used to determine which leg the mouse prefers to use to assess limb function (fig. 39). Mice were allowed to acclimate to the chamber on at least 3 occasions before taking measurements. Mice were advanced within the chamber to stand with 1 hind paw on each scale. The weight placed on each hind limb was measured over a period of 3 seconds. At each time point, at least 3 independent measurements were made for each animal. The results were expressed as the percentage of weight placed on the operative limb to the contralateral non-operative limb. As shown in fig. 40, untreated mice that had undergone osteoarthritis surgery prefer to use the non-operated hind limb over the post-operative hind limb (Δ). However, clearing aged cells with Nutlin-3A abolished this effect in mice (v) that had undergone surgery.

The function of the limb was also assessed by hotplate (hotplate) analysis 4 weeks after surgery to illustrate the sensitivity and response to painful stimuli. Briefly, mice were placed on a hot plate at 55 ℃. When placed on a hot plate surface, mice will lift and lick their paw (lick paw response) due to reaching the pain threshold. The lag time of hindlimb reaction (licking paw reaction) was recorded as the reaction time. As shown in fig. 41, untreated mice that had undergone osteoarthritis surgery had increased response time compared to normal mice that were not surgically altered (■). However, treatment of mice that had undergone osteoarthritis surgery with Nutlin-3A decreased response time in a significant manner

Histopathology of osteoarthritis induced by ACL surgery showed that the proteoglycan layer was disrupted. Elimination of senescent cells by Nutlin-3A completely abolished this effect. The clearance of senescent cells from 3MR mice treated with GCV killed senescent cells had the same effect on the pathophysiology of osteoarthritis as Nutlin-3A. See fig. 42.

Example 22

Effect of senescent cell ablation in animal models of atherosclerosis

A schematic representation of two mouse models of atherosclerosis is presented in FIGS. 43A-B. The study shown in FIG. 43A evaluated senescent cells from LDLR by Nutlin-3A^-/-The extent to which clearance in the plaques of the mice reduced the plaque load. Starting at week 0 and throughout the study, two groups of LDLR were tested^-/-Mice (10 weeks) were fed a 42% calorie fat-derived High Fat Diet (HFD) (Harlan Teklad td.88137). For two groups of LDLR^-/-Mice (10 weeks) fed normal dietFood (-HFD). From week 0 to week 2, one group of HFD mice and-HFD mice was treated with Nutlin-3A (25mg/kg, intra-abdominal). The treatment cycle was 14 days treatment and 14 days no treatment. Vehicle was administered to one group of HFD mice and one group of-HFD mice. At week 4 (time point 1), a group of mice was sacrificed to evaluate the presence of senescent cells in the plaques. Nutlin-3A and vehicle administration were repeated from week 4 to week 6 for some of the remaining mice. At week 8 (time point 2), the mice were sacrificed to evaluate the presence of senescent cells in the plaques. From week 8 to 10, the remaining mice were treated with Nutlin-3A or vehicle. At week 12 (time point 3), the mice were sacrificed to assess the level of plaques and the number of senescent cells in the plaques.

At time point 1, LDLR fed HFD and treated with Nutlin-3A or vehicle, compared to mice fed HFD, was measured^-/-Plasma lipid levels in mice (n ═ 3 per group). Plasma was collected at around three pm (mid-afternoon) and analyzed for circulating lipids and lipoproteins. The data are shown in FIGS. 44A-D.

At the end of time point 1, the LDLR fed HFD and treated with Nutlin-3A or vehicle will be fed^-/-Mice were sacrificed (n-3, all groups) and aortic arch dissected for RT-PCR analysis of SASP factor and senescent cell markers. Values were normalized to GAPDH and expressed as age-matched, vehicle-treated LDLR relative to normal diet^-/-Fold change in mice. The data show that after 1 treatment cycle (see FIGS. 45A-D), LDLR on HFD was fed^-/-Depletion of senescent cells by Nutlin-3A in mice reduced the expression of several SASP factors and senescent cell markers (MMP3, MMP13, PAI1, p21, IGFBP2, IL-1A, and IL-1B).

At the end of time point 2, LDLR-^/Mice (n ═ 3 in all groups) were sacrificed and aortic arch dissected for RT-PCR analysis of SASP factor and senescent cell markers. Values were normalized to GAPDH and expressed as age-matched, vehicle-treated LDLR-^/Fold change in mice. Data show that in HFD miceExpression of some of the SASP factors and senescent cell markers in the aortic arch (fig. 46A-C). LDLR-^/Clearance of senescent cells by Nutlin-3A over multiple treatment cycles in mice reduced the expression of most markers (FIG. 46A-B).

At the end of time point 3, LDLR-^/Mice (n ═ 3 for all groups) were sacrificed and the aorta dissected and stained with sudan IV to detect the presence of lipids. The body composition of the mice was analyzed by MRI and circulating blood cells were counted by Hemavet. The data show that treatment with Nutlin-3A reduced plaque mass in the descending aorta by about 45% (fig. 47A-C). As shown in figures 48A-B, platelet and lymphocyte counts were equal in Nutlin-3A and vehicle-treated mice. Treatment with Nutlin-3A also reduced the mass and body fat composition of HFD-fed mice as shown in figures 49A-B.

The study shown in FIG. 43B evaluated the results from LDLR^-/-Acyclovir-based senescent cell clearance in/3 MR dual transgenic mice improved the extent of pre-existing atherosclerotic disease. Starting from week 0 until week 12, is LDLR^-/-/3MR double transgenic mice (10 weeks) and LDLR^-/-Single transgenic mice (10 weeks) were fed a high fat diet. Ganciclovir (25mg/kg, i.p.) was administered to mice in both groups from weeks 12 to 13 and weeks 14 to 15. At week 16, the level of plaques and the number of senescent cells in the plaques were determined. As shown in FIG. 50, with LDLR-^/mouse/HFD control (n ═ 9) LDLR-^/-/3MR double transgenic mice (n ═ 10) cleared senescent cells by GCV and decreased% of aorta covered by plaques. As shown in FIG. 51, with LDLR-^/Depletion of senescent cells by GCV also resulted in HFD-fed LDLR-^/Plaques in-/3 MR double transgenic mice (n ═ 3) decrease in cross-sectional area.

Example 23

Senescent cell clearance to maintain aged cardiac stress tolerance

To study the effects of senescent cell clearance on health and longevity, groups of INK-ATTAC transgenic mice were established in the context of FVB x 129Sv/E x C57BL/6 mixes or C57BL/6 pure genes. At 12 months of age, half of the mice in each group were injected three times a week with AP20187 to induce apoptosis of p 16-positive senescent cells (0.2 mg/kg and 2mg/kg AP20187 for the mixed and pure C57BL/6 group, respectively), while the other half of each group received vehicle. At 18 months of age, a subset of mice from each cohort of males and females were subjected to a cardiac stress test in which the mice were injected with a lethal dose of isoproterenol (680mg/kg) and the time to cardiac arrest was recorded. The 18-month old untreated (vehicle) mice consistently showed a significant acceleration of cardiac arrest compared to the 12-month old control mice, while the AP20187 treated mice maintained young cardioprotection against isoproterenol, regardless of gender and genetic background (see figure 52).

It is known that cardioprotective signaling pathways provide resistance to metabolic stresses such as ischemia and hypoxia decline (decline) (Granfeldt et al, 2009, cardiovasc. res. 83: 234-. However, cardioprotective signaling degrades with aging, thus decreasing the function and adaptive reserve capacity of the heart (Ogawa et al, 1992, Circulation 86: 494-. The ATP-dependent K channel (KATP) plays a central role in cardioprotective signaling (Gross and Auchampach, 1992, Cardiovasc. Res.26: 1011-. These KATP channels are composed of pore-forming subunit Kir6.2/Kir6.1, regulatory subunit Sur2a and other accessory proteins. The KATP channel is thought to decline with aging due to decreased expression of Sur2a (Du et al, 2006, FASEB J. 20: 1131-. It has been shown that an increase in the expression of Sur2a by dietary changes (Sukhodub et al, 2011, J.cell. mol. Med. 15:1703-1712) or by the transgenic pathway (Sudhir et al, 2011, biogentology 12:147-155) maintains cardiac stress tolerance in aged mice. Thus, the contribution of senescent cells to the age-related decline in Sur2a expression was examined when 18-month-old AP 20187-treated and vehicle-treated mice were subjected to the aforementioned cardiac stress test. Indeed, the young performance of isoproterenol stress test in 18-month-old AP 20187-treated females correlated consistently with sustained Sur2a expression (see fig. 53). Taken together, these experiments indicate that the presence of senescent cells with aging adversely affects KATP pathway function, and senescent cell clearance is an effective therapy to counteract this degeneration. Sustained cardiac performance may contribute to the median lifespan extension observed in AP 20187-treated INK-ATTAC mice.

Example 24

Elimination of senescent cells improves atherosclerosis in LDLR-/-/3 MR mice

In LDLR-^/The effect of senescent cell clearance on the stability and size of mature atherosclerotic plaques was studied in/3 MR double transgenic mice. For LDLR starting from 10 weeks of age, starting from week 0 to week 12.5^-/-/3MR double transgenic mice (10 weeks) and LDLR^-/-Single transgenic mice (control) were fed a 42% calorie fat-derived high fat diet (Harlan Teklad td.88137) and mice were returned to normal diet after 12.5 weeks. On the next 100 days after week 12.5, two groups of mice were treated with ganciclovir, wherein each treatment cycle contained 5 days of ganciclovir (25mg/kg daily, i.v.) and 14 days without treatment. At the end of the 100 day treatment period, mice were sacrificed, plasma and tissues were collected and the amount of atherosclerosis would be measured.

Descending aorta was dissected and stained with sudan IV to visualize plaque lipids. As shown in FIGS. 54A-B, LDLR-^/-/3MR double transgenic mice compared with HFD fed LDLR-^/Control mice had fewer atherosclerotic plaques and lower staining intensity. As measured by area of Sudan IV staining, with LDLR-^/Ganciclovir-treated LDLR-^/The% of aorta covered in plaques was also significantly lower in/3 MR mice (see fig. 54C).

LDLR-^/Control mice and LDLR-^/Plaques of-/3 MR mice (see dotted circle plaques in FIGS. 55A-B, respectively) were cut into cross-sections and stained to characterize the totality of atherosclerotic plaquesAnd (5) constructing. "#" indicates fat located on the outer side of the aorta (see fig. 55A). Plaques labeled with "", and "", respectively, in fig. 55A and B are shown in stained cross-sections in fig. 55B and D, respectively. As shown in FIGS. 55B and D, with LDLR-^/Ganciclovir treated LDLR-^/-/3 clearance of senescent cells in MR mice has an effect on plaque morphology. The plaques from the control mice accumulate within recognizable "lipid pockets". LDLR-^/Plaques of-/3 MR mice show the presence of a thick fibrin cap and the absence of a lipid pocket. The destruction or tearing of the cap of the lipid-rich plaque is the trigger for a coronary event by exposure of the plaque thrombotic components to platelets and the thrombogenic components of blood. Plaques that grow faster due to rapid lipid deposition and have a thinner fibrin cap are prone to rupture. Slow growing plaques with mature fibrin caps tend to stabilize and do not easily rupture. Taken together, these experiments indicate that removal of senescent cells can affect the formation of atherosclerotic plaques and have a stabilizing effect.

Tissue sections of atherosclerotic plaques were prepared and stained to detect SA- β -GAL. X-GAL crystals are located in lysosomes of lipid-bearing macrophage foam cells and smooth muscle foam cells (see FIGS. 56-58).

Example 25

Effect of senescent cell clearance in Lung disease model

An animal model study evaluated the effect of senescent cell clearance in the transgenic mouse line 3MR with bleomycin-induced lung injury. In the bleomycin lesion model of idiopathic pulmonary fibrosis, mice develop pulmonary fibrosis within 7-14 days after bleomycin treatment (see, e.g., Limjunyawong et al 2014, physical Reports2: e 00249; Daniels et al 2004, J.Clin.invest.114: 1308-. Bleomycin (2.5U/kg bleomycin in 50 μ l PBS) was administered to anesthetized 6-8 week old 3MR mice by intratracheal inhalation using a microsprayer syringe (Penn-Century, Inc.) as described by Daniels et al (2004, J.Clin. Invest.114: 1308) with the use of a microsprayer syringe (Penn-Century, Inc.). Salt administration to control miceAnd (3) water. The day after bleomycin treatment, Ganciclovir (GCV) (25mg/kg in PBS) was administered. 3MR mice were treated via intraperitoneal injection of ganciclovir for 5 consecutive days, followed by a rest for 5 days, followed by a treatment cycle for 5 consecutive days. Untreated mice received an equal volume of vehicle. Lung function was assessed by monitoring oxygen saturation on days 7, 14 and 21 after bleomycin treatment using a mouses stat physiosuite pulse oxygen saturation meter (Kent Scientific). Animals were anesthetized with isoflurane (1.5%) and toe clips were applied. Mice were monitored for 30 seconds and mean peripheral capillary oxygen saturation (SpO) calculated during this period₂) And (6) measuring the values. As shown in figure 59, bleomycin administration significantly reduced SpO in vehicle-treated mice₂Horizontal, and removal of senescent cells results in higher SpO₂Levels which are close to normal levels at 21 days after bleomycin administration. Mice were tested for Airway Hypersensitivity (AHR) 21 days after bleomycin treatment. AHR was measured in mice by methacholine challenge, while other parameters of lung function (airway mechanics, lung volume and lung compliance) were determined using the SCIREQ flexiVent ventilator. When under ketamine/xylazine anesthesia and undergoing tracheal cannulation via tracheostomy (19Fr blunt Luer cannula), mice were evaluated for airway resistance (elasticity) and compliance (compliance) at baseline and in response to increasing concentrations of methacholine (0 to 50mg/ml in PBS) delivered via nebulization (AeroNeb) as described by aravacutan et al (am.j. physiol.lung cell.mol.physiol. (2012)303: L669-L681). The animals were maintained at 37 ℃ and simultaneously under muscle paralysis (pantocrine bromide); by using FlexiVent encapsulated in Stabile 8^TMVentilator and pulmonary mechanics systems (SCIREQ, Montreal, Quebec, Canada) measure airway function. As shown in figure 60A, bleomycin administration increased lung elasticity while ganciclovir treatment decreased lung elasticity in vehicle-treated mice. As shown in fig. 60B-C, bleomycin administration reduced static and (dynamic) compliance in vehicle-treated mice. Clearance of senescent cells with ganciclovir significantly improved the compliance values in bleomycin-exposed mice (fig. 60B-C). Although not statistically significant because the animal groups were too small in size, the data indicateElimination of senescent cells with senolytic agent (Nutlin-3A) also reduced lung elasticity and increased lung compliance in bleomycin-exposed mice. Mice were euthanized by intraperitoneal injection of pentobarbital. Bronchoalveolar lavage (BAL) fluid and lungs were obtained and analyzed. Lung hydroxyproline content was measured and histopathology was quantified as described by Christensen et al (1999, am. J. Pathol. 155: 1773-1779). RNA was extracted from lung tissue to measure senescent cell markers in treated and control mice by qRT-PCR.

The effect of eliminating senescent cells in the bleomycin-induced lung injury model of IPF was also studied in INK-ATTAC transgenic mice in the study design described above. INK-ATTAC (by targeting p16 which activates caspase enzymes^Ink4aApoptosis) transgenic mice with a p16 gene^Ink4aFK 506-binding protein (FKBP) -caspase 8(Casp8) fusion polypeptides under promoter control (see, e.g., Baker et al, Nature, supra; International patent application publication No. WO/2012/177927). In the presence of AP20187, a synthetic drug that induces dimerization of membrane-bound tetradecylated FKBP-Casp8 fusion protein, via p16^Ink4aSenescent cells with promoters specifically expressing the FKBP-Casp8 fusion protein undergo programmed cell death (apoptosis) (see, e.g., Baker, Nature, supra, in fig. 1).

The second study also evaluated the effect of senescent cells cleared using senolytic agents in C57BL6/J mice with bleomycin-induced lung injury. Bleomycin was administered to 6 week old C57BL6/J mice as described above. Senolytic agents are administered during the first and third weeks after bleomycin treatment. Control mice were treated with vehicle. Clearance of senescent cells and lung function/histopathology were assessed 21 days after bleomycin treatment.

In a second animal model of lung disease (e.g., COPD), mice are exposed to cigarette smoke. The effect of senolytic agents on mice exposed to smoke was assessed by senescent cell clearance, lung function and histopathology.

Six-week-old 3MR (n ═ 35) or INK-ATTAC (n ═ 35) mice were exposed chronically to the strain Teague TE-10In the production of cigarette smoke, the system is an automatically controlled smoking machine that produces a combination of sidestream and mainstream cigarette smoke in a compartment, the combination of smoke being delivered to a collection and mixing compartment where different amounts of air are mixed with the smoke mixture. The COPD protocol was adapted from the COPD core facility of John Hopkins university (Internet site jhu. edu/Biswal/expose _ core/COPD. html # Cigarette _ Smoke) (Rangasamy et al, 2004, J.Clin. Invest.114: 1248-. Mice received a total of 6 hours of cigarette smoke exposure per day for 5 days per week over 6 months. Each lit cigarette (3R4F study cigarette each cigarette contained 10.9mg Total Particulates (TPM), 9.4mg tar and 0.726mg nicotine, and 11.9mg carbon monoxide [ University of Kentucky, Lexington, KY ]]) Puff for 2 seconds and a total of 8 puffs per minute to provide 35cm³Wherein the flow rate is 1.05L/mi. The smoker was adjusted to produce a mixture of sidestream smoke (89%) and mainstream smoke (11%) by smoldering 2 cigarettes at a time. Monitoring the total suspended particles (80-120 mg/m) in the air of the smoke compartment³) And carbon monoxide (350 ppm). Beginning on day 7, the (10) INK-ATTAC and (10) 3MR mice were treated with AP20187(3 times per week) or more loxivir (continuous 5 days treatment followed by 16 days withdrawal, repeated until the end of the experiment), respectively. An equal number of mice received the corresponding vehicle. The remaining 30 mice (15. sup. INK-ATTAC and 15. sup. 3MR) were evenly grouped, with 5 of each genetically modified strain placed into three different treatment groups. One group (n-10) received Nutlin-3A (25mg/kg in 10% DMSO/3% Tween (Tween) -20 in PBS for 14 consecutive days followed by 14 days off dosing and repeated until the end of the experiment). One group (n ═ 10) received ABT-263(Navitoclax) (100mg/kg dissolved in 15% DMSO/5% tween-20, treatment continued for 7 days, followed by 14 days of discontinuation, repeated until the end of the experiment), and the last group (n ═ 10) received only vehicle for ABT-263 (15% DMSO/5% tween-20) following the same treatment protocol as ABT-263. Another 70 animals that did not receive exposure to cigarette smoke were used as controls for the experiment.

Two months after cigarette smoke exposureThereafter, lung function was assessed by monitoring oxygen saturation using a mouses stat physiosuite pulse oxygen saturation meter (Kent Scientific). Animals were anesthetized with isoflurane (1.5%) and toe clips were applied. Mice were monitored for 30 seconds and mean peripheral capillary oxygen saturation (SpO) calculated during this period₂) And (6) measuring the values. As shown in FIG. 61, elimination of senescent cells in mice via AP20187, ganciclovir, ABT-263(Navi) or Nutlin-3A after 2 months of cigarette smoke exposure resulted in SpO₂The level was statistically significantly increased compared to the untreated control.

At the end of the experimental period, mice were examined for methacholine-stimulated Airway Hyperresponsiveness (AHR) using the SCIREQ flexiVent ventilator and pulmonary mechanics system as described above. Following AHR measurement, mice were killed by intraperitoneal injection of pentobarbital as previously described to analyze lung pathology in depth (Rangasamy et al, 2004, j.clin.invest.114: 1248-. Briefly, lungs were inflated with 0.5% low melting agarose at a constant pressure of 25 cm. A portion of lung tissue was collected to extract RNA and qRT-PCR analysis was performed on the aging markers. The other parts of the lung were fixed in 10% buffered formalin and embedded in paraffin. Sections (5 μm) were stained with hematoxylin and eosin. The mean alveolar diameter, alveolar length and mean linear intercept were determined by computer-assisted morphometry using Image Pro Plus software (MediaCybernetics).

The potential therapeutic effect of eliminating senescent cells after complete COPD formation can be assessed in 3MR or INK-ATTAC mice. Six-week-old 3MR or INK-ATTAC mice were exposed to cigarette smoke for a long period of 6 months as described above. 3MR or INK-ATTAC mice were treated with ganciclovir (5 consecutive days of treatment followed by 16 days of withdrawal) or AP20187(3 times/week), respectively, 6 months after smoke exposure began, until 9 months after smoke exposure began, at which time aged cell clearance, lung function and histopathology were assessed.

Example 26

In vitro cell assay for determining senolytic activity of MDM2 inhibitor RG-7112

The lung fibroblast cell line IMR90 (human primary lung fibroblasts,CCL-186TM, Manassas, Virginia) were seeded in six-well plates and senescence was induced with 10Gy of Ionizing Radiation (IR). The senescent phenotype was allowed to develop for at least 7 days.

After the senescent phenotype was established, cells were re-seeded into 96-well plates and senescent cells (irradiated) and non-senescent cells (non-irradiated) were exposed to eight two-fold series of dilutions of the MDM2 inhibitor RG-7112 (see structure in figure 62A) starting at 100 μ M for a period of 3 or 6 days. After three days, use is made of a commercially available productThe Cell Viability was determined by luminescennt Cell Viability Assay (Promega Corporation, Madison, Wisconsin). The assay determines the number of viable cells in a culture based on quantifying the presence of ATP, which is an indicator of metabolically active cells. Figure 62 shows IMR90 cell viability after 123 days (see figure 62B) and six days (see figure 62C) exposure to RG-71123.

Example 27

Effect of senescent cell depletion by ABT-263 on reducing chemotherapy-related side effects

The ability of senolytic agents such as ABT-263 to reduce chemotherapy-related side effects such as fatigue was examined in p16-3MR transgenic mice. In addition to doxorubicin, cellular senescence was also induced when paclitaxel was administered to animals. See example 2 for a description of the p16-3MR transgenic mouse model.

Paclitaxel induced senescence and SASP in p16-3MR transgenic mice. Groups of mice were treated with 20mg/kg paclitaxel or vehicle three times every two days (n-4). The aging of paclitaxel-treated mice was observed by luminescence as shown (see fig. 63A). Aiming at the target gene: each of p16, the 3MR transgene and IL-6, measured the levels of mRNA in the skin. As shown in figure 63B, mRNA levels were increased for each of p16, 3MR, and IL-6 in paclitaxel-treated animals compared to vehicle-treated animals.

Fig. 64 shows a schematic of this experiment. In this experiment, paclitaxel was administered to a group of p16-3mr mice (n ═ 4) three times every two days. Two days after the third dose of paclitaxel, ganciclovir was administered intraperitoneally at 25mg/kg for three days per day (days 1, 2 and 3). ABT-263(100mg/kg) was administered intraperitoneally daily for seven days following paclitaxel administration. Two days after the last dose of ABT-263, all groups of animals were placed in metabolic cages (promethion, able system interpositional, Las Vegas, NV) to monitor spontaneous activity as determined by spinning wheel counts. Data were collected and analyzed two days later. Data are shown in figure 64 (left). Clearance of senescent cells with ABT-263 and ganciclovir restored approximately 70% of the rotawheel count reduction resulting from chemotherapy treatment.

Example 28

Chemotherapy medicine for inducing aging

To examine the senescence induced by different chemotherapeutic drugs, thalidomide (100 mg/kg; 7 injections per day); romidepsin (1 mg/kg; 3 injections); pomalidomide (5 mg/kg; 7 injections per day); lenalidomide (50 mg/kg; 7 injections per day); 5-azacytidine (5 mg/kg; 3 injections) groups of p16-3MR animals (n-4) were treated and compared to dorbixin (10mg/kg, 2-4 injections over 7 days). The luminescence levels in the drug-treated animals are shown in fig. 65. Treatment of animals with pomalidomide (omalidomide), lenalidomide and doxorubicin resulted in significant levels of senescent cells (p < 0.05).

Example 29

Pathways associated with senescence

Proteomic analysis of lysates of aged or non-aged human abdominal subcutaneous preadipocytes was performed by nano LC MS/MS. Preadipocytes, one of the most abundant cell types susceptible to aging in humans, were extracted from adipose tissue of nine different healthy kidney transplant donors by collagenase digestion. The consent of the donor was obtained beforehand. Senescence was induced by 10Gy radiation or by serial subculture. Bioinformatics methods are used to identify pathways susceptible to existing drugs and capable of mediating cell death.

Use of senescence-associated beta-galactoseGlycosidase (SA-. beta.gal) activity the percentage of senescent cells present in irradiated cell cultures was assessed. To be considered a senescent culture in this experiment, 75% or more of the cells were required to exhibit SA- β gal activity. Whole cell lysates and cell supernatants were collected. Proteins were separated on 1D SDS-PAGE. Sections of the gel were destained, reduced, alkylated and digested with trypsin. In THERMO SCIENTIFIC^TMThe extracted peptides were analyzed by nano-LC-MS/MS on a Q active mass spectrometer. Proteins were identified and quantified using LC prognesis software (Nonlinear Dynamics, UK). The data was then submitted to Ingeneity, Metacore, Cytoscope and other software for pathway and protein network analysis. Among the pathways that are altered during aging are those involved in cell survival signaling and inflammatory pathways. These pathways include at least PI3K/AKT, Src kinase signaling, insulin/IGF-1 signaling, p38/MAPK, NF-. kappa.B signaling, TGF β signaling, and mTOR/protein translation.

FIG. 66 shows confirmatory (confirmatory) Western immunoblots of proteins involved in these and related pathways at various times (24 hours; 3, 6, 8, 11, 15, 20 and 25 days) after irradiation. Phosphorylated polypeptides in senescent Cell samples were detected using horseradish peroxidase-labeled antibodies (Cell Signaling Technology, Danvers, MA) specific for the polypeptides shown in figure 66. Senescence was fully established between day 25 and day 30 in these cells.

Example 30

Reduction of high fat feeding induced senescence by senolytic agents in P16-3MR mice

A group of p16-3MR mice was fed with a high fat diet (60% fat) or a regular food diet (n ═ 6) for four month mice. The presence of senescent cells (i.e., p16 positive cells) was determined by measuring luminescence. As shown in figure 67, animals fed a high fat diet had an increased number of senescent cells compared to animals fed a regular diet.

The animals were then treated with ganciclovir or vehicle to determine whether depletion of senescent cells reduced the presence of senescent cells in adipose tissue. Groups of animals were treated with ganciclovir or vehicle. Ganciclovir (25mg/kg) was administered daily for five consecutive days. The presence of senescent cells in perirenal (perirenal), epididymal or subcutaneous inguinal adipose tissue was detected by SA- β -Gal staining. Data were analyzed by ANOVA. The results are shown in fig. 68. A significant reduction in the presence of senescent cells in epididymal fat was observed. p ═ 0.004.

Example 31

Clearance of senescent cells improves glucose tolerance and insulin sensitivity.

A group of p16-3MR mice was fed with a high fat diet or regular food diet for four month mice (n ═ 9). The animals were then treated with ganciclovir (3 rounds of 25mg/kg ganciclovir daily for five consecutive days) or vehicle. Glucose boluses were given at time zero and blood glucose was monitored at 20, 30, 60 and 120 minutes after glucose delivery to determine glucose utilization (see figure 69A). This was also quantified as the "area under the curve" (AUC) (see fig. 69B and 69C), with higher AUC values indicating glucose intolerance. The AUC of ganciclovir-treated mice was significantly lower than the vehicle-treated counterpart, but not as low as the food-fed animals. Hemoglobin A1C was lower in ganciclovir-treated mice (see fig. 69C), indicating that the animals had improved longer-term glucose treatment as well.

Insulin sensitivity was also determined (insulin tolerance test (ITT)). The results are shown in fig. 70. After administration of the glucose bolus at time zero, ganciclovir-treated mice at 0, 14, 30, 60 and 120 minutes showed greater blood glucose reduction (see fig. 70A), indicating that senescent cell clearance improves insulin sensitivity. No change was observed in the insulin tolerance test when ganciclovir was administered to wild-type mice (see figure 70B).

Changes in body weight, body composition and food intake were also monitored. Body composition or food intake (measured in grams per week) as monitored by fat percentage without change in body weight by ganciclovir treatment.

Example 32

Senolytic activity of BCL-2/BCL-XL inhibitors

Cell viability assay was used to assess cell viability following a-1155463 treatment. A general timeline and procedure for the cell count assay is shown in fig. 18 and described in example 7. IMR90 cells (human primary lung fibroblasts) were seeded in six-well plates and cell senescence was induced with 10Gy of Ionizing Radiation (IR) (day 0). The medium was refreshed every 3 days. The senescent phenotype was allowed to develop for 7 days, at which time point cell counts were performed to determine the baseline number of cells, which were then plated into 96-well plates. On day 8, senescent cells (irradiated) and non-senescent cells (non-irradiated) were exposed to the serial dilutions of A-1155463 for a period of 24 hours. Each case was inoculated in triplicate. Use of(CTG) luminescence Cell Viability Assay determines Cell survival. The assay determines the number of viable cells in the culture medium based on the quantification of ATP present, which is an indicator of metabolically active cells.

FIG. 71 shows the IC50 profile of A-1155463 in senescent and non-senescent cells. The IC50 curve is a graph of the percentage of cells surviving after treatment.

The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the application data sheet, including U.S. provisional patent serial No. 61/932,704 filed on month 1, 28, 2014; 61/932,711 filed on 28 days 1 month 2014; 61/979,911 filed 4, 15, 2014; 62/002,709 filed 5/23/2014; 62/042,708 filed on day 27 of 8 months 2014; 62/044,664 filed on 9/2/2014; 62/057,820 filed on 30/9/2014; 62/057,825 filed on 30/9/2014; 62/057,828 filed on 30/9/2014; 62/061,627 filed on 8/10/2014; and 62/061,629 filed on 8/10/2014, which are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified, if necessary, to employ concepts of the various patents, applications and publications to provide yet further embodiments.

These and other changes can be made to the described embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

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<212> DNA

<213> Artificial sequence

<220>

<223> shRNA sequences

<400> 7

ccgggctcac tcttcagtcg gaaatctcga gatttccgac tgaagagtga gctttttg 58

<210> 8

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> shRNA sequences

<400> 8

gctcactctt cagtcggaa 19

<210> 9

<211> 58

<212> DNA

<213> Artificial sequence

<220>

<223> shRNA sequences

<400> 9

ccgggtggaa ctctatggga acaatctcga gattgttccc atagagttcc actttttg 58

<210> 10

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> shRNA sequences

<400> 10

gtggaactct atgggaaca 19

<210> 11

<211> 58

<212> DNA

<213> Artificial sequence

<220>

<223> shRNA sequences

<400> 11

ccgggtttag tgatgtggaa gagaactcga gttctcttcc acatcactaa actttttg 58

<210> 12

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> shRNA sequences

<400> 12

gtttagtgat gtggaagag 19

<210> 13

<211> 58

<212> DNA

<213> Artificial sequence

<220>

<223> shRNA sequences

<400> 13

ccgggctcac tcttcagtcg gaaatctcga gatttccgac tgaagagtga gctttttg 58

<210> 14

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> shRNA sequences

<400> 14

gctcactctt cagtcggaaa t 21

<210> 15

<211> 58

<212> DNA

<213> Artificial sequence

<220>

<223> shRNA sequences

<400> 15

ccggtggcag actttgtagg ttatactcga gtataaccta caaagtctgc catttttg 58

<210> 16

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> shRNA sequences

<400> 16

tggcagactt tgtaggtta 19

<210> 17

<211> 58

<212> DNA

<213> Artificial sequence

<220>

<223> shRNA sequences

<400> 17

ccgggtcaac aaggagatgg aaccactcga gtggttccat ctccttgttg actttttg 58

<210> 18

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> shRNA sequences

<400> 18

gtcaacaagg agatggaac 19

<210> 19

<211> 58

<212> DNA

<213> Artificial sequence

<220>

<223> shRNA sequences

<400> 19

ccggcagaag ggttatgtct gtggactcga gtccacagac ataacccttc tgtttttg 58

<210> 20

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> shRNA sequences

<400> 20

cagaagggtt atgtctgtg 19

<210> 21

<211> 59

<212> DNA

<213> Artificial sequence

<220>

<223> shRNA sequences

<400> 21

ccggccatta gatgagtggg atttactcga gtaaatccca ctcatctaat ggttttttg 59

<210> 22

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> shRNA sequences

<400> 22

ccattagatg agtgggattt a 21

Claims (44)

1. A unit dose of a pharmaceutical composition comprising an amount of a compound of formula (I):

wherein the composition contains a formulation of the compound suitable for administration to an osteoarthritic joint in a subject in need thereof; and wherein the formulation of the composition and the amount of the compound in the unit dose configure the unit dose to effectively treat the osteoarthritic joint without causing adverse effects by reducing the severity of one or more symptoms or signs of osteoarthritis in the joint when the joint is administered as a single dose.

2. The unit dose of claim 1, wherein the pharmaceutical composition contains hyaluronic acid.

3. The unit dose of claim 1, wherein the formulation is a sustained release formulation comprising a gel, a polymer matrix, or microparticles.

4. The unit dose of claim 1, which is effective to reduce the severity of the one or more symptoms when administered as a single dose to the osteoarthritic joint followed by a non-treatment interval of at least two weeks.

5. The unit dose of claim 1, which is effective to inhibit erosion of the proteoglycan layer in an osteoarthritic joint when administered to a joint.

6. A product comprising a unit dose of the pharmaceutical composition of claim 1 and package insert describing the use and concomitant benefits of the composition in the treatment of osteoarthritis.

7. A medical device or apparatus containing a unit dose of the pharmaceutical composition of claim 1, wherein the device or apparatus is configured for injection of the pharmaceutical composition it contains into an osteoarthritic joint.

8. The unit dose according to claim 1, wherein one of the symptoms of reduced severity as a result of treatment with the unit dose is pain experienced in the osteoarthritic joint of the subject.

9. A method of treating an osteoarthritic joint in a subject comprising administering to the joint a pharmaceutical composition comprising a senolytic agent and a pharmaceutically compatible excipient such that the senolytic agent contacts and eliminates senescent cells in or around the joint, thereby alleviating a symptom of osteoarthritis in the joint, wherein the senolytic agent is a means of inhibiting MDM 2.

10. The method of claim 9, wherein the means of inhibiting MDM2 is an imidazoline compound.

11. The method of claim 10 wherein the imidazoline compound is 4- (4S, 5R) -4, 5-bis (4-chlorophenyl) -4, 5-dihydro-2- [ 4-methoxy-2- (1- (methylethoxy) phenyl ] -1H-imidazol-1-yl ] carbonyl ] -2-piperazinone or a pharmaceutically acceptable salt thereof (Nutlin-3 a).

12. The method of claim 9, wherein the MDM2 is inhibited by a means selected from the group consisting of Nultin-1, Nutlin-2, RG 7112, RG7388, DS-3032b, MI-63, MI-126, MI-122, MI-142, MI-147, MI-219, MI-220, MI-221, MI-773,3- (4-chlorophenyl) -3- ((1- (hydroxymethyl) cyclopropyl) methoxy) -2- (4-nitrobenzyl) isoindolin-1-one, Trametan, AM-8553, CGM097, R0-2443, R0-5963,

5- [ (3S) -3- (4-chlorophenyl) -4- [ (R) -1- (4-chlorophenyl) ethyl]-2, 5-dioxo-7-phenyl-1, 4-diAza derivatives-1-yl]The content of the valeric acid is shown in the specification,

5- [ (3S) -7- (2-bromophenyl) -3- (4-chlorophenyl) -4- [ (R) -1- (4-chlorophenyl) ethyl]-2, 5-dioxo-1, 4-diaza-1-yl]The content of the valeric acid is shown in the specification,

TDP521252, TDP665759, NSC279287 and pharmaceutically acceptable salts thereof.

13. A method of treating osteoarthritis comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I):

wherein R is selected from saturated and unsaturated 5-and 6-membered rings containing at least one heteroatom selected from S, N and O and optionally substituted by a group selected from lower alkyl, cycloalkyl, C ═ O-R1, hydroxy, lower alkyl substituted by lower alkoxy, lower alkyl substituted by NH2, lower alkyl substituted by C ═ O-R1, N-lower alkyl, -SO₂CH₃Substituted with O and CH2C OCH 3;

r1 is selected from the group consisting of hydrogen, lower alkyl, NH2, N-lower alkyl, lower alkyl substituted with hydroxy, lower alkyl substituted with NH2, and a 5-or 6-membered saturated ring containing at least one heteroatom selected from S, N and O;

x1 and X2 are each independently selected from the group consisting of hydrogen, lower alkoxy, -CH2OCH3-, -CH2OCH2CH3-, -OCH2CF3 and-OCH 2CH 2F; and

y1 and Y2 are each independently selected from-Cl, -Br, -NO2, -C.ident.N and-C.ident.CH.

14. The method of claim 13, wherein R is piperazin-2-one.

15. The method of claim 13, wherein Y1 and Y2 are both chloro.

16. The method of claim 13, wherein X1 and X2 are-O-CH- (CH3)2 and-O-CH 3, respectively.

17. The method of claim 13, wherein the compound is administered intra-articularly to the osteoarthritic joint of the subject.

18. The method of claim 17, wherein the symptoms of osteoarthritis experienced in the joint of the subject are alleviated.

19. The method of any one of claims 14-18 wherein the compound is 4- [ [ (4S, 5R) -4, 5-bis (4-chlorophenyl) -4, 5-dihydro-2- [ 4-methoxy- ]2- (1-methylethoxy) phenyl ] -1H-imidazol-1-yl ] carbonyl ] -2-piperazinone.

20. A method of selectively ablating senescent cells to alleviate symptoms of a pulmonary disease or disorder that is not cancer in a subject, the method comprising:

a therapeutically effective course of treatment of administering a pharmaceutical composition comprising an arylsulfonamide that selectively inhibits Bcl-xL in or around the lungs of a subject in need thereof;

wherein the pharmaceutical composition is formulated such that the arylsulfonamide contacts senescent cells located in or around the lung that cause symptoms of the lung disease or disorder,

wherein senescent cells are defined as non-cancerous cells expressing p16,

wherein the effective treatment course comprises:

(1) a treatment period in which the subject is administered one or more doses of the pharmaceutical composition to contact and selectively eliminate senescent cells from the lungs, and then

(2) For a treatment period of at least two months during which the pharmaceutical composition is not administered, and wherein symptoms of the pulmonary disease or disorder in the subject are alleviated as a result of the administration of one or more doses of the pharmaceutical composition during the treatment period selectively eliminating senescent cells from the lung.

21. The method of claim 20, wherein the arylsulfonamide includes a structure shown in formula (II):

wherein X3 is Cl or F;

x4 is azepan-1-yl, morpholin-4-yl, 1, 4-oxepan-4-yl, pyrrolidin-1-yl, N (CH3)2, N (CH3) (CH (CH3)2), 7-azabicyclo [2.2.1] hept-1-yl or 2-oxa-5-azabicyclo [2.2.1] hept-5-yl, R0 is

Wherein X5 is CH2, C (CH3)2 or CH2CH 2; x6 and X7 are both hydrogen or both methyl; x8 is F, Cl, Br or I; or either

X4 is azepan-1-yl, morpholin-4-yl, pyrrolidin-1-yl, N (CH3) (CH (CH3)2) or 7 azabicyclo [2.2.1] hept-1-yl, R0 is

；

Or either

X4 is N (CH3)2 or morpholin-4-yl, R0 is

22. The method of claim 20, wherein the arylsulfonamide is ABT-737 or ABT-263(Navitoclax), or a pharmaceutically acceptable salt thereof.

23. The method of claim 20, wherein the arylsulfonamide is administered as an aerosol.

24. A method of treating a pulmonary disease or disorder that is not cancer in the lungs of a subject in need thereof, the method comprising:

a course of treatment of administering to the subject a compound that constitutes a means of selectively inhibiting Bcl-2 or Bcl-xL in a pharmaceutical composition,

wherein the pharmaceutical composition is formulated such that the compound contacts p 16-positive aging cells located in or around the lung that promote symptoms of a pulmonary disease or disorder, thereby selectively eliminating these cells and alleviating these symptoms;

wherein the course of treatment comprises:

(1) a treatment period wherein the compound is administered into the lungs of the subject through the pulmonary airways such that the compound selectively removes senescent cells from the lungs, wherein senescent cells are defined as not p16 positive cells. Cancer cells; secondly, the

(2) A treatment period of at least two weeks during which no compound is administered and symptoms of the pulmonary disease or disorder are alleviated as a result of administration of the compound to the lung during the treatment period.

25. The method of claim 24, wherein the means for selectively inhibiting Bcl-2 or Bcl-xL is ABT 263(Navitoclax), or a pharmaceutically acceptable salt thereof.

26. The method as claimed in claim 24, wherein the means for selectively inhibiting Bcl-2 or Bcl-xL is selected from WEHI-539, A-1155463, ABT-737, ABT-199, Obatoclax, BXI-61, BXI-72,2,3-DCPE, the compound "21" (R) -4- (4-chlorophenyl) -3- (3- (4- (4- ((4- (dimethylamino) -1- (phenylthio) but-2-yl) amino) -3-nitrophenylsulfonylamino) phenyl) piperazin-1-yl) phenyl) -5-ethyl-1-methyl-1H-pyrrole-2-carboxylic acid, the compound 14 "(R) -5- (4-chlorophenyl) -4- (3- (4- (4- (4- ((4- (dimethylamino) -1- (phenylsulfanyl) but-2-yl) amino) -3-nitrophenylsulfonylamino) phenyl) piperazin-1-yl) phenyl) -1-ethyl-2-methyl-1H-pyrrole-3-carboxylic acid, "compound 15" (R) -5- (4-chlorophenyl) -4- (3- (4- (4- (4-) (-4- (dimethylamino) -1- (phenylthio) but-2-yl) amino) -3-nitrophenylsulfonylamino) phenyl) piperazin-1-yl) phenyl) -1-isopropyl-2-methyl-1H-pyrrole-3-carboxylic acid, BM-957, BM-1074, BM-1197 and pharmaceutically acceptable salts thereof.

27. The method of claim 24, wherein the pulmonary disease or disorder is Idiopathic Pulmonary Fibrosis (IPF), Chronic Obstructive Pulmonary Disease (COPD), or is caused or exacerbated by smoking.

28. The method of claim 24, which has the effect of increasing the mean peripheral capillary oxygen saturation (SpO2) in the subject.

29. A method of selectively removing senescent cells from the lungs of a subject in need thereof, the method comprising:

(1) administering a compound of an effective pharmaceutical formulation to the lungs via the pulmonary airways, contacting senescent cells in the lungs of the subject with a senolytic compound, thereby selectively promoting apoptosis of the cells; then the

(2) Waiting for a period of at least one month before administering more of the senolytic compound to the lung;

wherein the senolytic compound is a means of selectively inhibiting Bcl-2 or Bcl-xL; and

wherein the senescent cells are defined as p16 positive cells that are not cancer cells.

30. The method of claim 29, wherein the means for selectively inhibiting Bcl-2 or Bcl-xL is ABT-263(Navitoclax), or a pharmaceutically acceptable salt thereof.

31. A method of treating an ophthalmic disease or disorder in a subject, comprising:

administering to the eye or periocular of a subject in need thereof a pharmaceutical composition comprising an effective amount of a small molecule that selectively inhibits Bcl-2 or Bcl-xL,

thereby killing senescent cells in the eye that cause symptoms of the ophthalmic disease or disorder,

wherein senescent cells are defined as non-cancerous cells expressing p16,

the pharmaceutical composition is administered intraocularly or intravitreally, and

the pharmaceutical composition is administered in a therapeutically effective course of treatment comprising a treatment period followed by a non-treatment interval of at least two weeks.

32. The method of claim 31, wherein the ophthalmic disease or disorder is presbyopia, macular degeneration, or glaucoma.

33. The method of claim 31, wherein the means for selectively inhibiting Bcl-2 or Bcl-xL is selected from the group consisting of WEHI-539, A-1155463, ABT-737, ABT-199, Obatoclax, BXI-61, BXI-72,2,3-DCPE,

((R) -4- (4-chlorophenyl) -3- (3- (4- (4- (4- ((4- (dimethylamino) -1- (phenylthio) butane-20-) amino) -3-nitrophenylsulfonylamino) phenyl) piperazin-1-yl) phenyl) -5-ethyl-1-methyl-1H-pyrrole-2-carboxylic acid ("Compound 21"),

(R) -5- (4-) chlorophenyl) -4- (3- (4- (4- (4- ((4- (dimethylamino) -1- (phenylsulfanyl) but-2-yl) amino) -3-nitrophenylsulfonylamino) phenyl) piperazin-1-yl) phenyl) -1-ethyl-2-methyl-1H-pyrrole-3-carboxylic acid ("Compound 14"),

(R) -5- (4-chlorophenyl) -4- (3- (4- (4-) (4- (4- (dimethylamino) -1- (phenylsulfanyl) but-2-ylamino) -3-nitrophenylsulfonylamino) phenyl) piperazin-1-yl) phenyl) -1-isopropyl-2-methyl-1H-pyrrole-3-carboxylic acid ("Compound 15"),

BM-957, BM-1074 and BM-1197.

34. The method of claim 31, wherein the means for selectively inhibiting Bcl-2 or Bcl-xL is ABT-263 (Navitoclax).

35. The method of claim 31, wherein a single dose of the pharmaceutical composition is administered during the treatment.

36. A method of treating an ophthalmic disease or disorder that is not cancer in a subject,

the method comprises administering to the eye or around the eye of the subject a pharmaceutical composition comprising an effective amount of a compound that inhibits Bcl-2 and Bcl-xL in a pharmaceutically acceptable excipient,

wherein the compound is (R) -N- (4- (4- (3- (2- (4-chlorophenyl) -1-isopropyl-5-methyl-4- (methylsulfonyl) -1H-pyrrol-3-yl) -5-fluorophenyl) piperazin-1-yl) phenyl) -4- ((4- (4-hydroxypiperidin-1-yl) -1- (phenylthio) but-2-yl) amino) -3- ((trifluoromethyl) sulfonyl) benzenesulfonamide (BM 1197) or a salt thereof.

37. The method of claim 36, wherein the method is a method for preventing or delaying vision loss.

38. The method of claim 36, wherein the composition is administered to the eye by intravitreal injection.

39. A method of treating an ophthalmic disease or disorder in a subject not suffering from cancer,

the method comprises intraocular administration to the eye of a subject suffering from the disease or condition of an effective amount of:

40. a method of removing senescent cells from the eye of a subject in need thereof,

wherein senescent cells are defined as p16 positive cells that are not cancer cells,

the method comprises contacting senescent cells in the eye with an effective amount of (R) N (4- (3- (2- (4-chlorophenyl) -1-isopropyl-5-methyl-4- (methylsulfonyl) -) 1H-pyrrol-3-yl) -5-fluorophenyl) piperazin-1-yl) phenyl) -4- ((4- (4-hydroxypiperidin-1-yl) -1- (phenylthio) but-2-yl) amino) -3- ((trifluoromethyl) sulfonyl) benzenesulfonamide or a pharmaceutically acceptable salt thereof.

41. A method of inhibiting the progression of atherosclerosis in a subject comprising treating the subject with a course of treatment with a pharmaceutical composition comprising an amount of a senolytic compound preparation,

wherein the senolytic compound constitutes a means of selectively inhibiting the mouse double minute 2 homolog (MDM2),

wherein the course of treatment comprises:

(1) a treatment period during which the composition is systemically administered to the subject such that the compound contacts and selectively removes senescent cells from atherosclerotic plaques; followed by

(2) A treatment period of at least two weeks during which the composition is not administered to the subject, and the progression of atherosclerosis is inhibited as a result of the administration of the composition to the subject during the treatment period.

42. The method of claim 41, wherein the amount of the compound, the formulation of the composition, and the length of the treatment period are effective to delay thinning of the coating of the atherosclerotic plaque during the treatment period.

43. A method of removing senescent foam cell macrophages from the vasculature of a subject in need thereof, the method comprising:

treating a subject with a course of treatment with a pharmaceutical composition comprising a senolytic compound, the pharmaceutical composition comprising an amount of a preparation of the senolytic compound effective to eliminate senescent foam macrophages in the vasculature of the subject,

wherein the senolytic compound constitutes a means of selectively inhibiting the mouse double minute 2 homolog (MDM2),

wherein the course of treatment comprises:

(1) a treatment period during which the composition is systemically administered to the subject such that the compound selectively removes the aged foam macrophages from the vasculature of the subject; followed by

(2) A treatment period of at least two weeks during which the composition is not administered to a subject and occlusion of the vasculature due to plaque distribution or rupture is inhibited as a result of administration of the composition to the subject during the treatment period.

44. The method of claim 43 wherein the compound is (4- [ (4S, 5R) -4,5 bis (4-chlorophenyl) -4, 5-dihydro-2- [ 4-methoxy-2 (1 methylethoxy) phenyl ] ] -1H imidazol-1 yl ] carbonyl ] -2-piperazinone) (Nutlin 3A), or a pharmaceutically acceptable salt thereof.